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WO1992005261A1 - Improvements in or relating to organic compounds - Google Patents

Improvements in or relating to organic compounds Download PDF

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Publication number
WO1992005261A1
WO1992005261A1 PCT/EP1991/001786 EP9101786W WO9205261A1 WO 1992005261 A1 WO1992005261 A1 WO 1992005261A1 EP 9101786 W EP9101786 W EP 9101786W WO 9205261 A1 WO9205261 A1 WO 9205261A1
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Prior art keywords
wunl
promoter
dna
gene
plants
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PCT/EP1991/001786
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French (fr)
Inventor
Jürgen Logemann
Jeff Schell
Barbara Siebertz
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO1992005261A1 publication Critical patent/WO1992005261A1/en

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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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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 DNA sequence, an anther-specific promoter and the use thereof.
  • wunl gene A gene with a DNA sequence called "wunl gene", which is isolated from Solanu tuberosum, is known from literature references such as The Plant Cell, Vol. 1, 1989, p. 151-158; Proc. Natl. Acad.Sci.USA, Vol. 85, p. 1136-1140, Fbr.1988, as well as DE-OS 38 37 752. It is known that the wunl gene leads to the expression of gene products in the case of wounding or a pathogenic infection.
  • wunl gene a promoter region of 1022 bp length (+178bp wunl 5'untranslated region) maintains its wound- inducible activity even when the actual structural gene region is replaced by another structural gene (e.g. CAT, GUS). In this way, vound-inducible wunl promoter activity could be detected in leaf, stem and root of transgenic tobacco plants.
  • transgenic plants carrying fragments of the 5' region of the wound-inducible wunl gene in reverse orientation have constitutive anther-specific promoter activity.
  • bp are designated (-1022 +x)wunl (e.g. -571wunl).
  • Vectors having such promoter (e.g. -571wunl) attached at the 5'end of the GUS gene are designated (-1022 + x) wunl-GUS (e.g. -571 wunl-GUS).
  • DNA sequences of -1022 wunl cloned in reverse orientation are identified by r (e.g. 571r wunl resp. when cloned in front of the GUS reporter gene -571r wunl-GUS).
  • the invention therefore provides constitutive anther specific promoters, comprising a DNA fragment from the 5' region of the wunl gene, but which is in reverse orientation of said 5' region, DNA sequences homologous thereto, parts thereof or combinations of such parts.
  • vector as used herein relates to any vehicle by means of which DNA fragments can be introduced into a host organism.
  • the promoters of the invention are DNA fragments from the -1022 wunl promoter.
  • the DNA sequences of the invention have a bp range of from -571 of the wunl promoter to +178 of the 5' untranslated region of the wunl gene, or are homologous thereto, parts thereof or combinations of such parts, whereby the sequences are transcriptionally fused in the reverse orientation with gene.
  • a suitable example of such vector is a vector comprising the -571r wunl-GUS construct.
  • the DNA sequences of the invention have a bp range of from -571 to +1, or are homologous thereto, parts thereof or combinations of such parts and are transcriptionally fused in the normal or reverse orientation with a structural gene.
  • DNA sequence homologous to a DNA sequence of a constitutive anther specific promoter of the invention refers to a DNA sequence of a constitutive anther specific promoter of the invention wherein a number of nucleotides have been deleted and/or added but is still capable of hybridization to a nucleotide sequence having at least 50 nucleotides complementary to a DNA sequence of a constitutive anther specific promoter of the invention under appropriate hybridization conditions.
  • hybridization conditions conveniently include an incubation for 16 hours at 42°C, in a buffer system comprising 5 x standard saline citrate (SSC), 0.5 X sodium dodecylsulphate (SDS), 5 x Denhardt's solution, 50 X formamide and 100 ⁇ g/ml carrier DNA (hereinafter the buffer system), followed by washing 3 times with a buffer comprising 1 x SSC and 0.1 X SDS at 65 P C for approximately one hour each time.
  • SSC standard saline citrate
  • SDS sodium dodecylsulphate
  • 5 x Denhardt's solution 50 X formamide
  • carrier DNA hereinafter the buffer system
  • Preferred hybridization conditions for the purpose of the invention involve incubation in the buffer system for 16 hours at 49°C and washing 3 times with a buffer comprising 0.1 x SSC and 0.1 X SDS at 55°C for approximately one hour each time. Most preferred hybridization conditions for the purpose of the invention involve incubation in the buffer system for 16 hours at 55°C and washing 3 times with a buffer comprising 0.1 x SSC and 0.1 X SDS at 65°C for approximately one hour each time.
  • DNA sequences having constitutive anther specific promoter activity may be derived from the -1022wunl promoter by deleting fragments thereof, cloning it in reverse orientation and screening such shortened promoter for constitutive anther specific promoter activity in a manner known per se, e.g. employing a reporter gene such as GUS, analogous to the procedure described herein.
  • the DNA sequences according to the invention comprise at least 50 base pairs of the wunl promoter sequence in reverse orientation or a DNA sequence homologous thereto.
  • the DNA sequences according to the invention comprise at least 70 base pairs of the wunl promoter sequence in reverse orientation or a DNA sequence homologous thereto.
  • the DNA sequences according to the invention comprise at least 80 base pairs of the vunl promoter sequence in reverse orientation or a DNA sequence homologous thereto.
  • the promoters of the invention are useful, in that they allow the selective expression of gene products in the anthers.
  • a promoter of the invention is operably linked to a gene of which the selective expression of the gene product in the anthers is desired, e.g. to inhibit or influence cell development in the anthers or for ornamental purposes.
  • Such vectors also form part of the invention.
  • genes of which anther specific expression may be useful include genes causing development aberrations including male sterility, e.g. the rolC gene, or genes encoding gene products which inactivate the plant cell, e.g. the thionin gene, genes encoding gene products capable of degrading DNA, e.g. the gene encoding the synthesis of the Eco RI endonuclease or by inactivation of genes essential for anther development, e.g. the wunl antisense gene. Anther specific expression of such genes will result in male sterile plants. Where the gene resulting in male sterility is recessive, male sterile plants are obtained by selfing the plants containing the recessive gene.
  • a restorer system For the production of Fl hybrid seed the availability of a restorer system is necessary if the Fl hybrid crop involved is grown for its fruits or its seeds.
  • such restorer system can be made by using the anti-sense gene of the gene that was used to induce male sterility in the transgenic plant. Methods to achieve anti-sense inhibition are known to a person skilled in the art ⁇ ind include e.g.
  • the anti-sense rol C gene if the wunl-rolC construct was used to create male sterility; a similar strategy can be used for the thionine gene; in cases of the endonuclease gene induced male sterility not only the anti-sense strategy may be used, but also the insertion of the corresponding methylase gene creating a restorer for the endonuclease induced male sterility may be employed.
  • Male sterility brought about by gene technology has the advantage that by incorporating a definite gene into the genetic code of a plant, the remaining properties of the plant do not change. This applies especially to cultivated plants which genetically can be manipulated relatively well, e.g. to dicotyledoneous plants which are suited for tissue culture and transformable, but also to other cultivated plants. Male sterile ornamental plants have also the advantage that they may flower for a longer period of time.
  • the invention also provides male sterile plants or plant material comprising a constitutive anther specific promoter according to the invention.
  • promoters of the invention can be operably linked to transcription enhancers, such as the 35 S enhance .
  • Such plants or plant material are obtained in a manner known per se employing conventional transformation techniques, and where desired, followed by conventional breeding of thus obtained transgenic plants.
  • Fig. 1 Arrangement of wunl in the genomic clone wunl-85.
  • Fig. 2 Deletion analysis of the wunl-genomic clone wunl-85.
  • Fig. 3 Determination of the wunl transcription start point.
  • Fig. 4 Nucleotide sequence of wunl and flanking regions from wunl-85. CAAT-Box, TATA-Box and PolyA-signal are shown on black backgrounds. The start and stop of transcription are marked by arrows.
  • Fig. 5 Arrangement and position of important regions in the wunl gene.
  • Fig. 6 Construction of vunl-GUS 5' deletions for expression analysis in stable tobacco transformants. Between the left and the right T-DNA border sequence of the vector pPR69, a
  • NPT II gene lies under the control of the nopalin synthase promoter (nos). This is necessary for selection for kanamycin.
  • the GUS gene bears a nos-terminator sequence and is regulated by the various 5'deletion fragments of the wunl promoter. The end positions of the respective
  • the media used for the culture of bacteria are taken from data given by Maniatis et al. (1982).
  • the media employed are derived from the media (MS) given by Murashige and Skoog (1962).
  • Plasmids pUC8 (Vieira and Messing, 1982)
  • pPR69 a derivative of bin 19, Bevan 1984
  • Phages EMBL4 (Frischholz et al. , 1983)
  • RNA isolation of RNA from various organs of the potato and of tobacco are carried out as described by Logemann et al., (1987).
  • RNA is separated by electrophoresis on a 1.5% formaldehyde- agarose-gel (Lehrach et al. , (1977)). As described by Willmitzer et al., (1982), the RNA is subsequently transferred onto nitrocellulose, fixed and hybridized on - -? radioactively-labelled cDNA, washed and exposed.
  • Nuclear DNA is recovered by the method of Bedbrook (1981) from potato leaves and used for cloning in lambda phages EMBL4. DNA from transformed tissue is purified by combined lyses with Triton X 100, SDS, and proteinase K (Wassenegger, 1988). "Southern Blot" Analysis
  • DNA is separated by electrophoresis on 0.8 to 1.22 agarose gels, transferred onto nitrocellulose and fixed (Southern, 1975), also hybridized and washed as described by Willmitzer et al., (1981).
  • Genomic potato DNA is isolated by the method of Bedbrook, 1981 from leaf material of the haploid species AM 80/5793 and digested totally with EcoRI.
  • EMBL4 DNA is digested with EcoRI into three fragments, whereby the two vector arms are separated from the middle fragment by means of gel electrophoresis separation with subsequent fragment isolation.
  • commercially available, purified EMBL4 arms are also used (Amersham).
  • the ligated, high molecular weight DNA is packaged in vitro into phage heads (Hohn and Murray, 1977; Hohn, 1979) employing packaging material from a "Lambda in vitro packaging kit" from the company Amersham.
  • the genomic bank is plated out at a concentration of 25000 plaques per plate (25x25 cm).
  • a bacteria culture 20 ml of a bacteria culture, lysed with C600, are mixed with chloroform, in order to obtain a bacteria-free supernatant after subsequent centrifugation. Sedimentation of the phages from the supernatant is effected by centrifugation for 4 hours at 10000 rpm.
  • the phage sediment is taken up in 500 ul phage buffer (10 mM tris-HCl pH 8.0, 10 mM MgCl 2 ), and treated with DNase and RNase. After extraction of the DNA by means of phenolization several times, the phage-DNA is EtOH-precipitated, washed with 70 X EtOH and taken up in TE.
  • the fragment to be examined is cloned in M13mpl8 in both orientations. Since exonuclease III digests 5' projecting ends, but are spares 3' projecting ends, 20 ug of the DNA to be analysed are digested with two restriction enzymes which produce a 5' and a 3' end on one side of the cloned fragment. By successive stopping of this reaction, fragments which are 200 bp smaller are isolated, and the projecting ends thereof are transformed into ligatable smooth ends by means of subsequent Sl-treatment. Finally, transformation of this DNA into BMH 7118 cells takes place, with subsequent single-stranded DNA isolation.
  • Sequencing of single-stranded DNA is carried out by the chain- terminating method of Sanger et al., (1977). Separation of the reaction products is performed on 6X and QX sequencing gels (Ansorge and de Mayer, 1980; Ansorge and Barker, 1984). SI analysis
  • the starting point of transcription is effected according to Berk and Sharp (1987).
  • the 1.2 kb fragment is isolated from the plasmid pLSOOO with EcoRI and Xhol, and dephosphorylated by treatment with phosphatase. Then, radioactive labelling of the 5' OH ends takes place through the combination of polynucleotide kinase and gamma- 32 P-ATP. After denaturing of the DNA fragments, they are hybridized with 50 ug of whole RNA (hybridization buffer: 80% formamide, 0.4 M NaCl, 40 mM PIPES pH 6.4 and 1 mM EDTA.
  • RNA-DNA hybrids compared with DNA-DNA hybrids (Casey and Davidson, 1977).
  • the temperature at the start of hybridization starts at 80°C and is lowered over night to 40°C.
  • Sl-nuclease digestion 120 U/ml removes unpaired strands.
  • the radioactively labelled Xhol cutting site found in the 5' untranslated region of the wunl gene will be protected by its homology to the mRNA.
  • the SI protected DNA strand is separated by electrophoresis on a sequencing gel, whereby the sequence of a known DNA fragment is used for length comparison.
  • the DNA cloned in E. coli is transferred to A. tumefaciens LBA4404 (Hoeke a et al., 1983) by the method described by Van Haute et al., (1983), by conjugation .
  • the Agrobacterium strains containing the appropriate LBA4404 vector required for infection are grown in selective antibiotic medium (Zambrisky et al. , 1983), sedimented by centrifugation and washed in YEB medium without antibiotics. After further sedimentation and absorption in 3MS medium, the bacteria are used for infection.
  • Sterile leaf pieces of about 1 cm 2 size of tobacco species SNN and W38 are immersed into the Agrobacterium suspension described above, with subsequent transfer onto 3MS medium. After incubation for 2 days at 16 hours light and 25°C to 27°C, the pieces are transferred onto MSC16 for callus and shoot induction. Shoots appearing after 4-6 weeks are cut off and incubated on MSC15 medium.
  • the DNA from the plants is isolated by a varied method of Murray and Thompson (1980), cut with restriction enzymes and separated on a gel. After transfer of this DNA onto nitrocellulose, it is hybridized with a radioactively labelled sample, which indicates the presence of this specific DNA in the genome of the plant under examination. Detection of GUS activity
  • Plants regenerated from transformation are examined using the method of Jefferson (1987) for the degree of activity of the enzyme ⁇ -glucuronidase (GUS).
  • GUS ⁇ -glucuronidase
  • An additional histochemical test indicates localisation in the tissue.
  • thin sections of the plants are incubated with the substrate X-Gluc, 5-bromo-4-chloro-3-indolyl glucuronide. A blue precipitate forms at the site of enzyme activity.
  • the wunl-cDNA equivalent fragment in wunl-85 has a size of 4 Kb. Thus, it corresponds exactly to the fragment size of EcoRI digested DNA from the haploid potato, which hybridizes with wunl-cDNA. Based on the asymmetrical Xhol cutting site in the 5'region of the cDNA clone, and the use of radioactive cDNA samples which only cover the 5' region of the cDNA clone, the orientation of the gene in the 4 Kb fragment could be determined. Accordingly, 5' of the wunl gene there is a promoter region of ca. 1 Kb size, while at 3' from the gene there is a non-homologous part of ca. 2 Kb (fig. 5).
  • the EcoRI fragment of 8 Kb size additionally contain in wunl-85 could not be referred to for closer analysis of the wunl gene. Based on the digestion of the potato DNA with EcoRI, it is probable that during the subsequent ligation, two fragments not belonging together were brought into the EMBL4 vector, that is, neither can they enter a functional relationship with one another.
  • the genomic analysis of the wunl coding gene is carried out. Since in the restriction behaviour, no differences are established between wunl-85 and wunl-22, the 4 Kb fragment of wunl-85 is ligated into EcoRI cleaved pUC8 for further analysis (fig. 1). In order to sequence the wunl promoter and also the wunl gene, further re-cloning of the 4 Kb fragment into the Eco RI cutting site of M13mpl8 took place. Determination of its orientation is carried out using control digestion at the asymmetrical Xhol cutting site. The clones 85 * mpl8 and 85mpl8 represent both orientations of the fragment (fig. 2).
  • the method of SI nuclease mapping is used.
  • the principle of this method is based on the fact that as a result of hybridization of single-stranded DNA from the 5' region of the wunl gene with wunl-mRNA, only the regions which because of their homology may form a double strand are protected from degradation of the single-strand- specific nuclease SI.
  • the size of the protected DNA fragment can be determined on a sequencing gel and thus the start of transcription can be followed back.
  • a 1.2 Kb fragment is isolated from pLSOOO, and it contains the promoter and parts of the 5' untranslated region of the wunl gene.
  • This fragment is radioactively labelled with 32 P with polynucleotide kinase at the 5' end of the Xho I site, and hybridized on 50 ug of total RNA from wounded potato tubers.
  • the size of the protected fragment is determined on a sequencing gel; it varies between 162-178 bp (fig. 3).
  • the start of transcription begins 178 bp upstream from the Xho I site, i.e. with the sequence ACC ⁇ TAC.
  • This sequence conforms in the central region (CAT) with the consensus sequence, determined by Joshi et al., 1987, for the start of transcription CTCATCA. Further information is provided from the position of the start of transcription (figs. 4, 5):
  • CAAT box described by Benoist et al., 1980, in the region between -60 and -80 can be found in the wunl promoter at position -58 (CAAACT).
  • the 5' untranslated region of the wunl gene extends over 217 bp and is therefore comparatively large.
  • the wunl-mRNA coding gene thus encloses 794 bp, which corresponds very exactly with the size determination of wunl-mRNA based on "Northern-Blot" analysis.
  • the wunl gene has no introns at its disposal.
  • the promoter -1022 wunl is recloned into vector pPR69 (Fig. 6).
  • the deletion fragments beyond the Xhol cutting site are cut out of M13mpl8.
  • the promoter -1022 wunl and the promoter fragment -571 wunl were cloned in reverse orientation in front of the GUS gene (1022r; 571r). This was done by filling up the 5' overlapping ends of the fragments with DNA polymerase I and recloning it in the reversed orientation into the vector. Correct integration was tested by hybridisation with wunl radioactively labelled probes and by restriction analysis.
  • the plasmids pLS034-1022, -1022r and -571r are transformed into the agrobacterial strain LBA4404. These agrobacteria are then used for transformation of the tobacco cultivar Wisconsin 38 (W38) by means of the leaf section infection method. Under kanamycin selection, the callus produced on the plant are regenerated and are examined on Southern Blot for correct integration of the pLS034-DNA into the plant genome.
  • the functional analysis of the transgenic tobacco plants is extended to the detection of GUS activity in greenhouse plants.
  • Non-transformed tobacco plants serve as a control (K).
  • the activity of the enzyme in various tissues of the plant is analysed by means of histochemical in-situ analysis. For this, thin sections of the tissue are incubated over night at 37°C with the substrate of the enzyme, 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). A blue precipitate forms at the site of enzyme activity.
  • This analysis indicates that in plants of constructions pLS034, -571r and -1022r, no activity can be detected in leaves, stem or roots.
  • Plants carrying the full wunl promoter (-1022), in its natural orientation, in their construction show activity in all the said tissues.
  • Plants of construction pLS034-1022 show GUS activity in the stomium of the anthers and in the pollen grain. 30% of the pollen grain show blue colouring after incubation with X-Gluc. Anthers of pLS034-571r show the same pattern of activity. In control plants, as with plants of construction pLS034-1022r, no blue colouring is detected in the stomium, and colouring of the pollen grain is reduced to 0.75%.
  • Comparison of the measured GUS activity of anthers from the various constructions shows 2-10 times stronger activity of pLS034-1022 than in pLS034-571r.
  • the average enzyme activity of the full promoter in anthers reaches 1276 pmol MU/mg/min.
  • Construction pLS034-571r shows an average enzyme activity of 217 pmol MU/mg/min in the anthers.
  • the 35S promoter of cauliflower mosaic virus is active in anthers. Its activity is 500 pmol MU/mg/min. Since the 35S promoter of the CaMV is a strong promoter which is active in plants, the deletion fragments of the wunl promoter extend into this activity range throughout. Transformation of tobacco employing the rol C-gene
  • the purpose of the experiments was to obtain male sterility by targeting of rol C expression to anthers employing a -571r-rolC construct and a EN6-571r-rol C construct resp.
  • the -571r-rol C construct is the same construct as the -571r-GUS construct, except that the GUS has been replaced by the rol C-gene. Transformation with this construct results in male sterile plants.
  • the EN6-571r-rol C construct is a -571r-rol C construct having 5' thereof a fragment containing 6 times the 35 S enhancer (-96 to -420). Transformation with this construct results in male sterile plants.
  • Thionin is a small (5 Kd) protein which has been isolated from barley seeds and its activity of thionin is supposed to be similar to a detergent. Because of its bipolar character it is able to cause holes into membranes and therefore to destroy cells.
  • Thionin (5Kd) is synthesised as an inactive preprotein (15 Kd). It is flanked by a 3 Kd leader peptide at its N-terminus which is responsible for transport of the precursor protein to the endoplasmatic reticulum. At its C-terminus thionin is flanked by a 7 Kd acidic protein of unknown function.
  • thionin As a plant cell destroying agent the published sequencing data for hordothionin were used to synthesise an oligonucleotide coding for an active thionin.
  • Said "thionin” is translated as an active detergent directly into the cytoplasm employing a -571r-thionin construct.
  • wunl-gene is derived from potato plants, there is some cross homology with tobacco plants on Southern- and Northern- level. Anther-specific repression of wunl-gene products is sought employing the wunl-antisense constructs.

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Abstract

Promoters are described, which enable genes operably linked to them to be specifically expressed in anthers. By using anther-specific promoters, it is i.a. possible to obtain male sterility.

Description

IMPROVEMENTS IN OR RELATING TO ORGANIC COMPOUNDS
The invention relates to a DNA sequence, an anther-specific promoter and the use thereof.
The creation of cultivated plants with male sterility has been of economic interest for a long time, since this prevents self- pollination which proliferates among many plant species and hinders breeders when producing new cultivars.
Male sterility is of particular interest in the production of hybrid seeds. Many mono- and dicotyledoneous cultivated plants, such as cereals, vegetables and ornamentals, are sold as hybrid seeds. Production of these hybrid seeds is simplified considerably by the male sterility of one of the two parental lines. Some cultivated plants which have male sterility achieved by selection are already known. However, it is difficult to obtain such plants.
A gene with a DNA sequence called "wunl gene", which is isolated from Solanu tuberosum, is known from literature references such as The Plant Cell, Vol. 1, 1989, p. 151-158; Proc. Natl. Acad.Sci.USA, Vol. 85, p. 1136-1140, Fbr.1988, as well as DE-OS 38 37 752. It is known that the wunl gene leads to the expression of gene products in the case of wounding or a pathogenic infection. It could be shown that in the case of the "wunl gene" a promoter region of 1022 bp length (+178bp wunl 5'untranslated region) maintains its wound- inducible activity even when the actual structural gene region is replaced by another structural gene (e.g. CAT, GUS). In this way, vound-inducible wunl promoter activity could be detected in leaf, stem and root of transgenic tobacco plants.
It has also been reported by B. Siebertz et al (The Plant Cell - Vol. 1 (1989), 961-968) that certain 5' deletion fragments of the wunl promoter have constitutive promoter activity in anthers while essentially lacking wound- or infection-inducible activity in wounded or unwounded leaves, stems and roots, i.e. have constitutive anther-specific promoter activity. The range typical for the infection-induced DNA sequence of the wunl gene extends over 1201 base pairs (bp) (1022 bp of the wunl promoter + 178 bp of the 5'untranslated region). The anther-specific activity of certain fragments thereof was demonstrated with the GUS reporter gene, i.e. a gene producing a selectable or visibly screenable change of the phenotype in the transformed cell or plant, when attached to the shortened promoter.
Thus it was shown that transformation of tobacco plants with vectors comprising certain wunl promoter fragments transcriptionally fused with GUS, did not result in any detectable staining of leaves, stems, or roots but showed staining of the stomiu and pollen grains. As a control, tobacco plants were also transformed with a vector carrying the inverted wunl promoter (i.e. the wunl promoter in reverse orientation) transcriptionally fused with GUS. Thus, the latter transformed plants did not show any GUS activity.
It has now been found, that transgenic plants carrying fragments of the 5' region of the wound-inducible wunl gene in reverse orientation have constitutive anther-specific promoter activity.
In this way, if GUS is employed as reporter gene and for example 451 bp are removed at the 5'-end of the wunl promoter, this DNA fragment is cloned in reverse orientation in front of the GUS gene and tested e.g. in tobacco plants, said DNA sequence shows surprisingly a constitutive anther specific activity. For convenience, 1201 bp of the wunl gene consisting of the promoter region of 1022 bp and the 5' untranslated region of 178 bp attached at the 3'end of the promoter region are herein designated -1022 wunl promoter, or -1022 wunl. Fragments thereof shortened at the 5' end by x (e.g. 451) bp are designated (-1022 +x)wunl (e.g. -571wunl). Vectors having such promoter (e.g. -571wunl) attached at the 5'end of the GUS gene are designated (-1022 + x) wunl-GUS (e.g. -571 wunl-GUS). DNA sequences of -1022 wunl cloned in reverse orientation are identified by r (e.g. 571r wunl resp. when cloned in front of the GUS reporter gene -571r wunl-GUS).
The invention therefore provides constitutive anther specific promoters, comprising a DNA fragment from the 5' region of the wunl gene, but which is in reverse orientation of said 5' region, DNA sequences homologous thereto, parts thereof or combinations of such parts.
The term vector as used herein relates to any vehicle by means of which DNA fragments can be introduced into a host organism.
More particularly the promoters of the invention are DNA fragments from the -1022 wunl promoter. Preferably the DNA sequences of the invention have a bp range of from -571 of the wunl promoter to +178 of the 5' untranslated region of the wunl gene, or are homologous thereto, parts thereof or combinations of such parts, whereby the sequences are transcriptionally fused in the reverse orientation with gene. A suitable example of such vector is a vector comprising the -571r wunl-GUS construct. More preferably, the DNA sequences of the invention have a bp range of from -571 to +1, or are homologous thereto, parts thereof or combinations of such parts and are transcriptionally fused in the normal or reverse orientation with a structural gene.
The term DNA sequence homologous to a DNA sequence of a constitutive anther specific promoter of the invention refers to a DNA sequence of a constitutive anther specific promoter of the invention wherein a number of nucleotides have been deleted and/or added but is still capable of hybridization to a nucleotide sequence having at least 50 nucleotides complementary to a DNA sequence of a constitutive anther specific promoter of the invention under appropriate hybridization conditions. For the purpose of the invention appropriate hybridization conditions conveniently include an incubation for 16 hours at 42°C, in a buffer system comprising 5 x standard saline citrate (SSC), 0.5 X sodium dodecylsulphate (SDS), 5 x Denhardt's solution, 50 X formamide and 100 μg/ml carrier DNA (hereinafter the buffer system), followed by washing 3 times with a buffer comprising 1 x SSC and 0.1 X SDS at 65PC for approximately one hour each time.
Preferred hybridization conditions for the purpose of the invention involve incubation in the buffer system for 16 hours at 49°C and washing 3 times with a buffer comprising 0.1 x SSC and 0.1 X SDS at 55°C for approximately one hour each time. Most preferred hybridization conditions for the purpose of the invention involve incubation in the buffer system for 16 hours at 55°C and washing 3 times with a buffer comprising 0.1 x SSC and 0.1 X SDS at 65°C for approximately one hour each time.
Examples of constitutive anther specific promoters suitable for use in a vector according to the invention inlude the DNA sequences:
a) from +178 to -571 (i.e. -571r wunl) b) from +1 to -571 c) from +178 to -86 d) from +1 to -86
of the wunl-gene in the orientation as indicated (i.e. the reverse orientation of the wunl promoter), DNA sequences homologous thereto, and parts thereof or a combination of such parts.
Other DNA sequences having constitutive anther specific promoter activity may be derived from the -1022wunl promoter by deleting fragments thereof, cloning it in reverse orientation and screening such shortened promoter for constitutive anther specific promoter activity in a manner known per se, e.g. employing a reporter gene such as GUS, analogous to the procedure described herein.
Conveniently, the DNA sequences according to the invention comprise at least 50 base pairs of the wunl promoter sequence in reverse orientation or a DNA sequence homologous thereto. Preferably, the DNA sequences according to the invention comprise at least 70 base pairs of the wunl promoter sequence in reverse orientation or a DNA sequence homologous thereto. More preferably, the DNA sequences according to the invention comprise at least 80 base pairs of the vunl promoter sequence in reverse orientation or a DNA sequence homologous thereto.
The promoters of the invention are useful, in that they allow the selective expression of gene products in the anthers. For that purpose, a promoter of the invention is operably linked to a gene of which the selective expression of the gene product in the anthers is desired, e.g. to inhibit or influence cell development in the anthers or for ornamental purposes.
Such vectors also form part of the invention.
Examples of genes of which anther specific expression may be useful include genes causing development aberrations including male sterility, e.g. the rolC gene, or genes encoding gene products which inactivate the plant cell, e.g. the thionin gene, genes encoding gene products capable of degrading DNA, e.g. the gene encoding the synthesis of the Eco RI endonuclease or by inactivation of genes essential for anther development, e.g. the wunl antisense gene. Anther specific expression of such genes will result in male sterile plants. Where the gene resulting in male sterility is recessive, male sterile plants are obtained by selfing the plants containing the recessive gene. For the production of Fl hybrid seed the availability of a restorer system is necessary if the Fl hybrid crop involved is grown for its fruits or its seeds. For use in the present invention such restorer system can be made by using the anti-sense gene of the gene that was used to induce male sterility in the transgenic plant. Methods to achieve anti-sense inhibition are known to a person skilled in the art εind include e.g. the anti-sense rol C gene if the wunl-rolC construct was used to create male sterility; a similar strategy can be used for the thionine gene; in cases of the endonuclease gene induced male sterility not only the anti-sense strategy may be used, but also the insertion of the corresponding methylase gene creating a restorer for the endonuclease induced male sterility may be employed.
Combination of a genetically engineered male sterility in the mother line with a male line containing the appropriate restorer gene will result in a fertile Fl hybrid product capable of yielding normal amounts of fruits or seeds.
Male sterility brought about by gene technology has the advantage that by incorporating a definite gene into the genetic code of a plant, the remaining properties of the plant do not change. This applies especially to cultivated plants which genetically can be manipulated relatively well, e.g. to dicotyledoneous plants which are suited for tissue culture and transformable, but also to other cultivated plants. Male sterile ornamental plants have also the advantage that they may flower for a longer period of time.
It is of course possible to anther-specifically express genes which do not induce male sterility but have other properties of interest to breeders, e.g. gene products affecting the anthocyanine biosynthesis route to give coloured anthers in flowers having petals that have white or different colours. The latter possibility is of great interest for breeders of ornamentals. The invention also provides male sterile plants or plant material comprising a constitutive anther specific promoter according to the invention.
It will be appreciated that the promoters of the invention can be operably linked to transcription enhancers, such as the 35 S enhance .
Such plants or plant material are obtained in a manner known per se employing conventional transformation techniques, and where desired, followed by conventional breeding of thus obtained transgenic plants.
The following demonstrates that the vectors of the invention allow anther-specific expression of a gene.
In the figures are illustrated:
Fig. 1: Arrangement of wunl in the genomic clone wunl-85.
1.0 Kb of the wunl promoter, 0.8 Kb of the wunl gene coding region and 2.0 Kb of the 3' end are present on the 4 Kb EcoRI fragment of the genomic clone. This 4 Kb fragment is cloned into the EcoRI cutting site of pUC8 (pLSOOO).
Fig. 2: Deletion analysis of the wunl-genomic clone wunl-85.
On the basis of the asymmetrical Xhol cutting site in the genomic clone wunl-85, the 4 Kb fragment is detected in both orientations in M13mpl8 (85MP18; 85*MP18). DNA fragments of various sizes are obtained by successive digestion with exonucleaselll.
Fig. 3: Determination of the wunl transcription start point.
Wunl-mRNA from wounded potato tubers are hybridised against the 1.2 Kb EcoRI/XhoI Fragment of pLSOOO. This leads to a DNA-RNA hybrid which was protected against the single-strand-specific SI nuclease. As shown on a denaturing polyacrylamide-gel (lane TU) the length of the nuclease-protected DNA fragment is 162-178 bp. The actual start of transcription is thus 162-178 Bp 5' distant from the Xhol cutting site (A,C,G,T» sequence diagram to determine the size of the DNA fragment).
Fig. 4: Nucleotide sequence of wunl and flanking regions from wunl-85. CAAT-Box, TATA-Box and PolyA-signal are shown on black backgrounds. The start and stop of transcription are marked by arrows.
Fig. 5: Arrangement and position of important regions in the wunl gene.
The wunl promoter is marked in black, the wunl gene is marked in lines. Important recognition sequences are framed; the mRNA is indicated by a wavy line, and the protein-coding region by crosses. The size of the individual regions is given in (bp). Fig. 6: Construction of vunl-GUS 5' deletions for expression analysis in stable tobacco transformants. Between the left and the right T-DNA border sequence of the vector pPR69, a
NPT II gene lies under the control of the nopalin synthase promoter (nos). This is necessary for selection for kanamycin. The GUS gene bears a nos-terminator sequence and is regulated by the various 5'deletion fragments of the wunl promoter. The end positions of the respective
5'deletion fragments are given together with the respective construction. r = cloning of the promoter in reversed orientation.
Fig. 7: Tobacco leaves of transgenic plants from the greenhouse were analysed for their GUS-activity, depending on the various 5' promoter elements. The numbers on the ordinate correspond to the pLS034-delta-constructs shown on Fig. 6. K = untransformed tobacco plant. The unit pmol MU/mg protein/min on the abscissa indicates GUS-enzyme activity. The GUS-activity represents the average activity of non-wounded leaves out of 6-12 independent transgenic plants.
MATERIAL AND METHODS
Reference is made to the following literature:
(1) Ansorge, W., De Mayer, L. (1980).
Thermally stabilized very thin (0.02-0.3 mm) polyacrylamide gels for electrophoresis. J. Chromatogr., 202: 45-53.
(2) Ansorge, W., Barker, R. (1984).
System for DNA sequencing with resolution of up to 600 base pairs. J. Biochem.. Biophys. Meth., 9:33-47. (3) Bedbrook, J . (1981) .
A plant nuclear DNA preparation procedure. P.M.B. Newsletter II, 24.
(4) Benoist, C. , O'Hare, K. , Breathnach, R. , Chambon, P. (1980). The ovalbumin gene sequence of putative control regions. Nucl. Acids Res., 8:127-142.
(5) Benton, W.D. and Davis, R.W. (1977). Screening Lambda gt recombinant clones by hybridization to single plaques In situ. Science, 196:180.
(6) Berk, A. and Sharp, P.A. (1987). Spliced early mRNAs of SV40. Proc. Natl. Acad. Sci. USA, 75:1274-1278.
(7) Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12: 8711-8721.
(8) Ebert, P.R., HA, S.B., An, G. (1987). Identification of an essential upstream element in the nopalin synthase promoter by stable and transient assays. Proc. atl.Acad.Sci USA 84: 5745-5749.
(9) Fickett , F.W. ( 1982) .
Recognition of protein coding regions in DNA sequences. Nucl. Acids Res., 10:5303-5318.
(10) Frischauf, A.M., Lehrach, H., Poustka, A. and Murray, N. (1983). Lambda replacement vectors carrying polylinker sequences. J. Mol. Biol., 170:827-842.
(11) Henikoff, S. (1984).
Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene, 28:351-359.
(12) Hoekema, A., Hirsch, P., Hooykaas, P., Schilperoort, R. (1983). A binary plant vector strategy based on separation or vir- and T-region of A.tumefaciens. Nature 303: 179-180.
(13) Hohn, B. and Murray, K. (1977).
Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc. Natl. Acad. Sci. USA, 74: 3259-3263.
(14) Jefferson, R.A. (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol.Biol.Rep. 5: 387-405.
(15) Joshi, C.P. (1987). An inspection of the domain between putative TATA box and translation start side in 79 plant genes. Nucl. Acids Res. 15: 6643-6653.
(16) Lehrach, H., Diamond, D., Wozney, J.M. and Boedtker, H. (1977). RNA molecular weight determinations by gele electrophoresis under denaturing conditions, a critical re-examination. Biochemistry, 16:4743.
(17) Lipphardt, S. (1988).
Dissertation an der Universitat zu Kδln. (18) Logemann, J., Schell, J. and Willmitzer, L. (1987). Improved method for the isolation of RNA from plant tissues. Anal. Biochem., 163: 16-20.
(19) Maniatis, T., Fritsch, E.F., Sambrook, J. (1982). Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York.
(20) Messing, J., Geraghty, D., Hu, N.T., Kridl, J. and Rubenstein, I. (1977). Plant Gene Structure. In: Kosuge, F., Meredith, C.P., Hollaender, A. (eds.). Genetic engineering of plants. Plenum Press, New York: 211-227.
(21) Murashige, T., Skoog, F. (1962). A rapid method for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15:473-497.
(22) Murray, M.G., Thompson, W.F. (1980). Rapid isolation of high molecular weight plant DNA. Nucl.Acids.Res. 8: 4321-4325.
(23) Reiss, B., Sprengel, R., Will, H. , Schaller, H. (1984).
A new and sensitive method for qualitative and quantitative analysis of neomycinphosphotransferase in crude cell extracts. Gene 30:217-223.
(24) Sanger, F., Nicklen, S., Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74:5463-5467.
(25) Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98:503-517.
(26) Van Houte, E., Joos, H. , Maes, M. , Warren, G., Van Montagu, M., Schell, J. (1983). Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for reversed genetics of Ti-plasmids of Agrobacterium tumefaciens. EMBO J., 2:411-418.
(27) Vieira, J., Messing, J. (1982). The pUC plasmid, an M13mp7- derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene, 19:259-268.
(28) Wassenegger, M. (1988). Dissertation an der Universitat zu Kδln.
(29) Willmitzer, L. , Schmalenbach, W. and Schell, J. (1981). Transcription of T-DNA in octopine and nopaline crown gall tumours is inhibited by low concentration of alfa-amanitin Nucl. Acids Res., 9:19.
(30) Yanisch-Perron, C, Vieira, J., Messing, J. (1985).
Improved M13 phage cloning vectors and host strains: nucleotide sequencing of the M13mpl8 and pucl9 vectors. Gene 33: 103-119.
(31) Zambrisky, P., Joos, J., Genetello, C, Leemans, J., Van Montagu, M., Schell, J. (1983). Ti-Plasmid vector for the introduction of DNA into plant cells without alteration of their normal capacity. EMBO J., 1:147-152.
MATERIAL
Media
The media used for the culture of bacteria are taken from data given by Maniatis et al. (1982).
Plant media:
The media employed are derived from the media (MS) given by Murashige and Skoog (1962).
3MS : MS + 3 X saccharose
MSC15 MS + 2 X saccharose, 500 ug/ml claforan, 100 ug/ml kanamycin sulphate
MSC16 MS + 0.5 ug/ml BAP + 0.1 ug/ml NAA + 100 ug/ml kanamycin sulphate + 500 ug/ml claforan.
For solid medium, an additional 8 g/1 bacto-agar is added.
Strains and vectors
E. coli strains:
BMH 71-18: Δ (lac-proAB), thi, supE;
F'(lacli, ZdeltaM15, proA+B+) (Messing et al. 1977) C 600 : also known as CR34 (Maniatis et al., 1982) Agrobacteria strains: LBA 4404: (Hoekema et al., 1983)
Plasmids: pUC8 (Vieira and Messing, 1982) pPR69 (a derivative of bin 19, Bevan 1984) Phages: EMBL4 (Frischauf et al. , 1983)
M13mpl8 (Yanisch-Perron et al. , 1985)
Plants: Solanum tuberosum AM 80/5793 (haploid) Nicotiana tabacum Wisconsin 38 (W38)
METHODS
If not otherwise stated, all standard molecular-biological methods, e.g. restriction analysis, plasmid isolation, mini preparation of plasmid-DNA, transformation of bacteria etc., are carried out as described by Maniatis et al., (1982).
RNA isolation
Isolation of RNA from various organs of the potato and of tobacco are carried out as described by Logemann et al., (1987).
"Northern-Blot" Analysis
The RNA is separated by electrophoresis on a 1.5% formaldehyde- agarose-gel (Lehrach et al. , (1977)). As described by Willmitzer et al., (1982), the RNA is subsequently transferred onto nitrocellulose, fixed and hybridized on - -? radioactively-labelled cDNA, washed and exposed.
DNA isolation
Nuclear DNA is recovered by the method of Bedbrook (1981) from potato leaves and used for cloning in lambda phages EMBL4. DNA from transformed tissue is purified by combined lyses with Triton X 100, SDS, and proteinase K (Wassenegger, 1988). "Southern Blot" Analysis
DNA is separated by electrophoresis on 0.8 to 1.22 agarose gels, transferred onto nitrocellulose and fixed (Southern, 1975), also hybridized and washed as described by Willmitzer et al., (1981).
Establishment and research of a genomic bank
Isolation of genomic DNA
Genomic potato DNA is isolated by the method of Bedbrook, 1981 from leaf material of the haploid species AM 80/5793 and digested totally with EcoRI.
Isolation of the phages DNA
EMBL4 DNA is digested with EcoRI into three fragments, whereby the two vector arms are separated from the middle fragment by means of gel electrophoresis separation with subsequent fragment isolation. In addition, commercially available, purified EMBL4 arms are also used (Amersham).
Ligation and packaging
Following ligation of the EMBL4 arms with the EcoRI-digested genomic DNA, the ligated, high molecular weight DNA is packaged in vitro into phage heads (Hohn and Murray, 1977; Hohn, 1979) employing packaging material from a "Lambda in vitro packaging kit" from the company Amersham. The genomic bank is plated out at a concentration of 25000 plaques per plate (25x25 cm).
Plaque hybridization
The discovery of cDNA homologous lambda clones is made by means of plaque hybridization according to Benton and Davies (1977) using radioactively labelled cDNA. Hybridizing plaques are isolated and tested again. DNA preparation of recombinant phages
20 ml of a bacteria culture, lysed with C600, are mixed with chloroform, in order to obtain a bacteria-free supernatant after subsequent centrifugation. Sedimentation of the phages from the supernatant is effected by centrifugation for 4 hours at 10000 rpm. The phage sediment is taken up in 500 ul phage buffer (10 mM tris-HCl pH 8.0, 10 mM MgCl2), and treated with DNase and RNase. After extraction of the DNA by means of phenolization several times, the phage-DNA is EtOH-precipitated, washed with 70 X EtOH and taken up in TE.
Production of deletion mutants following exonuclease-III treatment (Henikoff et al., 1984)
The fragment to be examined is cloned in M13mpl8 in both orientations. Since exonuclease III digests 5' projecting ends, but are spares 3' projecting ends, 20 ug of the DNA to be analysed are digested with two restriction enzymes which produce a 5' and a 3' end on one side of the cloned fragment. By successive stopping of this reaction, fragments which are 200 bp smaller are isolated, and the projecting ends thereof are transformed into ligatable smooth ends by means of subsequent Sl-treatment. Finally, transformation of this DNA into BMH 7118 cells takes place, with subsequent single-stranded DNA isolation.
Sequencing
Sequencing of single-stranded DNA is carried out by the chain- terminating method of Sanger et al., (1977). Separation of the reaction products is performed on 6X and QX sequencing gels (Ansorge and de Mayer, 1980; Ansorge and Barker, 1984). SI analysis
Determination of the starting point of transcription is effected according to Berk and Sharp (1987). For this purpose, the 1.2 kb fragment is isolated from the plasmid pLSOOO with EcoRI and Xhol, and dephosphorylated by treatment with phosphatase. Then, radioactive labelling of the 5' OH ends takes place through the combination of polynucleotide kinase and gamma- 32P-ATP. After denaturing of the DNA fragments, they are hybridized with 50 ug of whole RNA (hybridization buffer: 80% formamide, 0.4 M NaCl, 40 mM PIPES pH 6.4 and 1 mM EDTA. This condition favours RNA-DNA hybrids compared with DNA-DNA hybrids (Casey and Davidson, 1977). The temperature at the start of hybridization starts at 80°C and is lowered over night to 40°C. Subsequent Sl-nuclease digestion (120 U/ml) removes unpaired strands. It is expected that the radioactively labelled Xhol cutting site found in the 5' untranslated region of the wunl gene will be protected by its homology to the mRNA. Finally, the SI protected DNA strand is separated by electrophoresis on a sequencing gel, whereby the sequence of a known DNA fragment is used for length comparison.
DNA transfer in agrobacteria
Transformation
The DNA cloned in E. coli is transferred to A. tumefaciens LBA4404 (Hoeke a et al., 1983) by the method described by Van Haute et al., (1983), by conjugation .
DNA analysis
Identification of DNA transfer to the Agrobacterium is effected by isolation of Agrobacterium DNA according to the method depicted by Ebert et al. (1987). Restriction cleavage of the DNA, the transfer to nitrocellulose and the hybridization on the corresponding radioactive sample provide evidence of the successful DNA transfer into the Agrobacterium. Transformation of tobacco
Culture of Agrobacterium
The Agrobacterium strains containing the appropriate LBA4404 vector required for infection are grown in selective antibiotic medium (Zambrisky et al. , 1983), sedimented by centrifugation and washed in YEB medium without antibiotics. After further sedimentation and absorption in 3MS medium, the bacteria are used for infection.
Infection of leaf sections
Sterile leaf pieces of about 1 cm2 size of tobacco species SNN and W38 are immersed into the Agrobacterium suspension described above, with subsequent transfer onto 3MS medium. After incubation for 2 days at 16 hours light and 25°C to 27°C, the pieces are transferred onto MSC16 for callus and shoot induction. Shoots appearing after 4-6 weeks are cut off and incubated on MSC15 medium.
Analysis of the transformed plants
Isolation of genoaic DNA
The DNA from the plants is isolated by a varied method of Murray and Thompson (1980), cut with restriction enzymes and separated on a gel. After transfer of this DNA onto nitrocellulose, it is hybridized with a radioactively labelled sample, which indicates the presence of this specific DNA in the genome of the plant under examination. Detection of GUS activity
Plants regenerated from transformation are examined using the method of Jefferson (1987) for the degree of activity of the enzyme β-glucuronidase (GUS). An additional histochemical test (Jefferson, 1987) indicates localisation in the tissue. For this purpose, thin sections of the plants are incubated with the substrate X-Gluc, 5-bromo-4-chloro-3-indolyl glucuronide. A blue precipitate forms at the site of enzyme activity.
RESULTS
Establishment of a genomic bank from a haploid potato
The small number of wunl genes in the genome of the haploid potato cultivar AM80/5793 led to the construction of a genomic bank consisting of genomic potato DNA which is digested with EcoRI.
10 ug of EcoRI cleaved DNA from AM80/5793 are ligated with EcoRI cleaved EMBL4 arms and plated on a C600 bacterial lawn. Ca. 500,000 plaques are obtained, which taken statistically, represents the genome of the potato.
Identification of wunl homologous EMBL4 clones
After the transfer of these plaques onto nitrocellulose, by using plaque hybridization techniques on radioactively labelled wunl- cDNA, two genomic clones (wunl-22 and wunl-85) are identified and purified.
Isolation of recombinant EMBL4 DNA from wunl-22, wunl-85, as well as their restriction mapping and hybridization on radioactive wunl- cDNA, give the following data:
The wunl-cDNA equivalent fragment in wunl-85 has a size of 4 Kb. Thus, it corresponds exactly to the fragment size of EcoRI digested DNA from the haploid potato, which hybridizes with wunl-cDNA. Based on the asymmetrical Xhol cutting site in the 5'region of the cDNA clone, and the use of radioactive cDNA samples which only cover the 5' region of the cDNA clone, the orientation of the gene in the 4 Kb fragment could be determined. Accordingly, 5' of the wunl gene there is a promoter region of ca. 1 Kb size, while at 3' from the gene there is a non-homologous part of ca. 2 Kb (fig. 5). The EcoRI fragment of 8 Kb size additionally contain in wunl-85 could not be referred to for closer analysis of the wunl gene. Based on the digestion of the potato DNA with EcoRI, it is probable that during the subsequent ligation, two fragments not belonging together were brought into the EMBL4 vector, that is, neither can they enter a functional relationship with one another.
Sequence analysis of the genomic clone wunl-85
In the following, the genomic analysis of the wunl coding gene is carried out. Since in the restriction behaviour, no differences are established between wunl-85 and wunl-22, the 4 Kb fragment of wunl-85 is ligated into EcoRI cleaved pUC8 for further analysis (fig. 1). In order to sequence the wunl promoter and also the wunl gene, further re-cloning of the 4 Kb fragment into the Eco RI cutting site of M13mpl8 took place. Determination of its orientation is carried out using control digestion at the asymmetrical Xhol cutting site. The clones 85*mpl8 and 85mpl8 represent both orientations of the fragment (fig. 2). Cutting of these plasmids with SphI and Xbal enables successive digestion, using exonucleaselll, of the 3' end of the wunl gene in the case of clone 85mpl8 and the 5' end of the wunl gene in the case of clone 85*mpl8 to take place.
Deletion clones resulting therefrom, with varying fragment sizes, are employed for sequencing. In all, it was possible to sequence the entire wunl gene bidirectionally, and additionally to analyse ca. 400 bp of the 3' end unidirectionally (fig. 4).
Determination of the transcription starting point
In order to identify the exact start of transcription of the wunl gene, the method of SI nuclease mapping is used. The principle of this method is based on the fact that as a result of hybridization of single-stranded DNA from the 5' region of the wunl gene with wunl-mRNA, only the regions which because of their homology may form a double strand are protected from degradation of the single-strand- specific nuclease SI. The size of the protected DNA fragment can be determined on a sequencing gel and thus the start of transcription can be followed back. Assisted by the cutting sites Xho I and Eco RI, a 1.2 Kb fragment is isolated from pLSOOO, and it contains the promoter and parts of the 5' untranslated region of the wunl gene. This fragment is radioactively labelled with 32P with polynucleotide kinase at the 5' end of the Xho I site, and hybridized on 50 ug of total RNA from wounded potato tubers. After SI treatment of the hybrid, the size of the protected fragment is determined on a sequencing gel; it varies between 162-178 bp (fig. 3). When starting from the longest fragment, the start of transcription begins 178 bp upstream from the Xho I site, i.e. with the sequence ACCΔTAC. This sequence conforms in the central region (CAT) with the consensus sequence, determined by Joshi et al., 1987, for the start of transcription CTCATCA. Further information is provided from the position of the start of transcription (figs. 4, 5):
1.) In position -33 viewed from the start of transcription, a TATA box CTATΔTATT is found, which conforms well with the consensus sequence TCACTATATATAG determined by Joshi et al., 1987.
2.) The CAAT box described by Benoist et al., 1980, in the region between -60 and -80 can be found in the wunl promoter at position -58 (CAAACT).
3.) The 5' untranslated region of the wunl gene extends over 217 bp and is therefore comparatively large.
4.) The wunl-mRNA coding gene thus encloses 794 bp, which corresponds very exactly with the size determination of wunl-mRNA based on "Northern-Blot" analysis.
5.) 97 bp of the 5' untranslated region in the wunl gene are missing in the cDNA clone wunl-25A2.
6.) Apart from the open reading frames already determined in the cDNA clone, no further branches can be found in the 5' untranslated region of the genomic clone.
7.) The wunl gene has no introns at its disposal. Establishment of the derivatives of the vector pPR69
The promoter -1022 wunl is recloned into vector pPR69 (Fig. 6). For preferential recloning, the deletion fragments beyond the Xhol cutting site are cut out of M13mpl8.
Additionally, the promoter -1022 wunl and the promoter fragment -571 wunl were cloned in reverse orientation in front of the GUS gene (1022r; 571r). This was done by filling up the 5' overlapping ends of the fragments with DNA polymerase I and recloning it in the reversed orientation into the vector. Correct integration was tested by hybridisation with wunl radioactively labelled probes and by restriction analysis.
Transformation of tobacco employing the GUS reporter gene
The plasmids pLS034-1022, -1022r and -571r are transformed into the agrobacterial strain LBA4404. These agrobacteria are then used for transformation of the tobacco cultivar Wisconsin 38 (W38) by means of the leaf section infection method. Under kanamycin selection, the callus produced on the plant are regenerated and are examined on Southern Blot for correct integration of the pLS034-DNA into the plant genome.
The functional analysis of the transgenic tobacco plants is extended to the detection of GUS activity in greenhouse plants.
Independent transfor ants of different constructions are analysed for their GUS activity in various tissues. Non-transformed tobacco plants serve as a control (K).
Comparison with the GUS activity in tobacco leaves shows little activity for plants of the construction pLS034-1022r, which is at about the level of activity of control plants. Compared with these, the GUS activity of pLS 034-571r is ca. 3-4 times higher, but substantially smaller than activity of the whole promoter (-1022) (pLS034-86), see fig. 7.
The activity of the enzyme in various tissues of the plant is analysed by means of histochemical in-situ analysis. For this, thin sections of the tissue are incubated over night at 37°C with the substrate of the enzyme, 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). A blue precipitate forms at the site of enzyme activity. This analysis indicates that in plants of constructions pLS034, -571r and -1022r, no activity can be detected in leaves, stem or roots.
Plants carrying the full wunl promoter (-1022), in its natural orientation, in their construction show activity in all the said tissues.
Another picture is seen during the analysis of flowers. In cross-sections of flowers of the various constructions, there was the following distribution of enzyme activity: Plants of construction pLS034-1022 show GUS activity in the stomium of the anthers and in the pollen grain. 30% of the pollen grain show blue colouring after incubation with X-Gluc. Anthers of pLS034-571r show the same pattern of activity. In control plants, as with plants of construction pLS034-1022r, no blue colouring is detected in the stomium, and colouring of the pollen grain is reduced to 0.75%.
Comparison of the measured GUS activity of anthers from the various constructions shows 2-10 times stronger activity of pLS034-1022 than in pLS034-571r. The average enzyme activity of the full promoter in anthers reaches 1276 pmol MU/mg/min. Construction pLS034-571r shows an average enzyme activity of 217 pmol MU/mg/min in the anthers.
As is known, the 35S promoter of cauliflower mosaic virus is active in anthers. Its activity is 500 pmol MU/mg/min. Since the 35S promoter of the CaMV is a strong promoter which is active in plants, the deletion fragments of the wunl promoter extend into this activity range throughout. Transformation of tobacco employing the rol C-gene
The purpose of the experiments was to obtain male sterility by targeting of rol C expression to anthers employing a -571r-rolC construct and a EN6-571r-rol C construct resp.
The -571r-rol C construct is the same construct as the -571r-GUS construct, except that the GUS has been replaced by the rol C-gene. Transformation with this construct results in male sterile plants.
The EN6-571r-rol C construct is a -571r-rol C construct having 5' thereof a fragment containing 6 times the 35 S enhancer (-96 to -420). Transformation with this construct results in male sterile plants.
Transformation of tobacco employing the thionin gene
Thionin is a small (5 Kd) protein which has been isolated from barley seeds and its activity of thionin is supposed to be similar to a detergent. Because of its bipolar character it is able to cause holes into membranes and therefore to destroy cells. Thionin (5Kd) is synthesised as an inactive preprotein (15 Kd). It is flanked by a 3 Kd leader peptide at its N-terminus which is responsible for transport of the precursor protein to the endoplasmatic reticulum. At its C-terminus thionin is flanked by a 7 Kd acidic protein of unknown function. In order to use thionin as a plant cell destroying agent the published sequencing data for hordothionin were used to synthesise an oligonucleotide coding for an active thionin. The thionin gene employed missed the leadersequence as well as the acidic protein and is hereinafter designated "thionin". Said "thionin" is translated as an active detergent directly into the cytoplasm employing a -571r-thionin construct.
Male sterile plants are obtained. Expression of wunl antisense:
Although the wunl-gene is derived from potato plants, there is some cross homology with tobacco plants on Southern- and Northern- level. Anther-specific repression of wunl-gene products is sought employing the wunl-antisense constructs.
-571r-5'antisense (wunl) and -571r-3'antisense (wunl) resp. Transgenic plants are regenerated and further tested.

Claims

CLAIMS: -
1. A constitutive anther specific promoter, characterised in that i comprises a DNA fragment from the 5' region of the wunl gene, which is in reverse orientation of said 5' region, DNA sequences homologous thereto, parts thereof or combinations of such parts, said DNA sequences having constitutive anther specific promoter activity.
2. The promoter according to Claim 1, characterised in that the DNA fragment is from the 1201 base pairs long DNA fraction consisting of the 1022 base pairs of the wunl promoter region and the 5' wunl untranslated region of 178 base pairs attached to the 3' end of the promoter region.
3. The promoter according to Claim 2, characterised in that the DNA fragment is from -571 of the wunl promoter to +178 of the 5' untranslated region of the wunl gene.
4. The promoter according to Claim 3, characterised in that the DNA fragment is from -571 of the wunl promoter to +1 of the 5' untranslated region.
5. A promoter comprising at least 50 base pairs of the DNA fragment according to Claim 3.
6. A promoter comprising at least 70 base pairs of the DNA fragment according to Claim 3.
7. A promoter comprising at least 80 base pairs of the DNA fragment according to Claim 3.
8. A promoter according to Claim 4, characterised in that the DNA fragment is from -86 of the wunl promoter to +1 of the 5' untranslated region.
9. A vector comprising the constitutive anther specific promoter of Claims 1 to 8 operably linked to a gene causing male sterility.
10. Plants or plant material containing the constitutive anther- specific promoter according to Claims 1 to 8.
PCT/EP1991/001786 1990-09-21 1991-09-19 Improvements in or relating to organic compounds WO1992005261A1 (en)

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WO1996040925A3 (en) * 1995-06-07 1997-05-01 Pioneer Hi Bred Int Regulatory elements conferring tapetum specificity

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO1994023043A3 (en) * 1993-03-31 1994-11-24 Nickerson Biocem Ltd Control of plant abscission and pod dehiscence
US5907081A (en) * 1993-03-31 1999-05-25 Biogemma Uk Limited Control of plant abscission and pod dehiscence
US5470359A (en) * 1994-04-21 1995-11-28 Pioneer Hi-Bred Internation, Inc. Regulatory element conferring tapetum specificity
US5837850A (en) * 1994-04-21 1998-11-17 Pioneer Hi-Bred International, Inc. Regulatory element conferring tapetum specificity
WO1996040925A3 (en) * 1995-06-07 1997-05-01 Pioneer Hi Bred Int Regulatory elements conferring tapetum specificity

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