AP181A - A DNA sequence, anther-specific promoter and the use thereof. - Google Patents

Abstract

DNA sequence having constitutive anther specific

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 selfpollination 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 dicotytedoneous 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 knovn. However, it is difficult to obtain such plants.

A gene with a DNA sequence called vunl gene, which is isolated from Solanum tuberosum, is knovn 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 veil as DE-OS 38 37 752. It is knovn that the vunl 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 vunl gene a promoter region of 1022 bp length (+179bp vunl 5'untranslated region) maintains its woundinducible activity even when the actual structural gene region is replaced by another structural gene (e.g. CAT, GUS). In this way,

BAD ORIGIN*¹- 2 Case 137-1046 vound-inducible vunl promoter activity could be detected in leaf, stem and root of transgenic tobacco plants.

In contrast to DE-OS 38 37 752, which is concerned with a woundstimulated DNA sequence and its use, the present invention relates to anther-specifically expressed DNA sequences and their use. DNA sequences were established, which enable structural genes to be expressed specifically in anthers.

It has been found that the wunl gene known from the references to literature already mentioned can not only be used as wound and/or infection-induced DNA sequence, but if the promoter of the wunl gene is shortened, an anther-specifically expressed DNA sequence may be attained. The range necessary to distinguish this feature extends over 1201 base pairs (bp) (1022 (bp) of the vunl promoter ·»· 179 bp of the 5'untranslated region). If the 1201 base pair length is reduced, e.g. by removal of 451 bp of the vunl promoter at the 5'end, this shortened promoter is only still active in the anthers. This can be proved vith a reporter gene, i.e. a gene producing a selectable or visibly screenable change of the phenotype in the transformed cell or plant, vhen attached to the shortened promoter. The change in the phenotype, e.g. colouration cannot be found in any part of the plant except for the pollen and stomium of anthers.

In this way, if GUS is employed as reporter gene and for example 451 bp ( -571vunl-GUS) are removed at the 5'-end of the vunl promoter, and this truncated promoter is tested e.g. in transgenic tobacco plants, there is no longer any activity in wounded or unvounded leaves, stems and roots.

The truncated promoter shows however surprisingly a constitutive activity in the anthers.

For convenience, 1201 bp of the vunl gene consisting of the promoter region of 1022 bp and the 5' untranslated region of 179 bp attachec at the 3'end of the promoter region is herein designated

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-1022 wunl. Fragments thereof shortened at the 5' end by x (e.g. 451) bp are designated (-1022 +x)vunl (e.g. -571vunl). Vectors having such promoter (e.g. -571vunl) attached at the 5'end of the US gene are designated (-1022 + x) wunl-GUS (e.g. -571 vunl-GUS).

The same expression behaviour observed with -571 vunl-GUS is also found vith other vunl promoter fragments, e.g. vith -86vunl-GUS.

The invention therefore provides a DNA sequence of a DNA fraction of the 5' upstream region of the vunl gene, DNA sequences homologous thereto and parts thereof, said DNA sequences having constitutive anther specific promoter activity.

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 vherein a number of nucleotides have been deleted and/or added but is still capable of hybridization to a nucleotide sequence 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^eC, 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 ug/ml carrier DNA (hereinafter the buffer system), followed by vashing 3 times vith a buffer comprising 1 x SSC and 0.1 X SDS at 65°C 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 vashing 3 times vith a buffer comprising 0.1 x SSC and 0.1 Z 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 vashing 3 times vith a buffer comprising 0.1 x SSC and 0.1 X SDS at 65^eC for approximately one hour each time.

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Suitable examples of promoter elements according to the invention having constitutive anther specific promoter activity are the base pair sequences

a)	from -571	to	+179,
b)	from -571	to	* 1,
c)	from - 86	to	+ 179,
d)	from - 86	to	+ 1,
e)	from -571	to -	111 3'linked	with	the -86	to +179 sequence
and f)	from -571	to -	111 3'linked	with	the -86	to +1 sequence,

of the vunl-gene, DNA sequences homologous thereto, and parts thereof.

Other DNA sequences having constitutive anther specific promoter activity may be derived from the -1022vunl promoter by eliminating base pairs thereof and screening such shortened promoter for competitive 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 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 structural 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.

The invention accordingly provides transformation vectors comprising a constitutive anther specific promoter according to the invention operably linked to a structural gene element.

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 , .- BAD ORIGINAL

- 5 Case 137-1046 inactivate the plant cell physiology, 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 vunl antisense gene. Anther specific expression of such genes will result in male sterile plants.

For the production of FI hybrid seed the availability of a restorer system is necessary if the FI 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 vas used to induce male sterility in the transgenic plant. Methods to achieve anti-sense inhibition are known to a person skilled in the art and could include e.g. the anti-sense rol C gene if the δ vunl-rolC construct vas 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 could be used, but also the insertion of the corresponding mechylase gene creating a restorer for the endonuclease Induced male sterility.

Combination of a genetically engineered male sterility in the mother line vith a male line containing the appropriate restorer gene will result in a fertile FI 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 vhich genetically can be manipulated relatively veil, e.g. to dicotyledoneous plants vhich are suited for tissue culture and transformable, but also to other cultivated plants.

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It is of course possible to anther-specifically express genes vhich 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.

Such plants or plant material may be 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 deletion of the wunl promoter, which is known from the above-mentioned references to literature, leads to anther-specific expression.

In the figures are illustrated:

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Fig. 1: Arrangement of vunl in the genomic clone vunl-85.

1.0 Kb of the vunl promoter, 0.8 Kb of the vunl 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 vas cloned into the EcoRI cutting site of pUC8 (pLSOOO).

Fig. 2: Deletion analysis of the vunl-genomic clone vunl-85.

On the basis of the asymmetrical Xhol cutting site in the genomic clone vunl-85, the 4 Kb fragment could be detected in both orientations in M13mpl8 (85MP18; 85*MP18). DNA fragments of various sizes vere obtained by successive digestion vith exonucleaselll.

Fig. 3: Determination of the vunl transcription start point.

Vunl-mRNA from vounded potato tubers vas hybridised against the 1.2 Kb EcoRI/XhoI Fragment of pLSOOO. This leads to a DNA-RNA hybrid vhich vas protected against the single-strand-specific SI nuclease. As shovn on a denaturing polyacrylamide-gel (lane TU) the length of the nuclease-protected DNA fragment is 162-179 bp. The actual start of transcription is thus 162-179 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 vunl and flanking regions from vunl-85. CAAT-Box, TATA-Box and PolyA-signal are shovn on black backgrounds. The start and stop of transcription are marked by arrows.

Fig. 5: Arrangement and position of important regions in the vunl gene.

The vunl promoter is marked in black, the vunl 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).

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Fig. 6:

Fig. 7:

Fig. 8:

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 vunl promoter. The end positions of the respective 5'deletion fragments are given together vith the respective construction.

R » cloning of the promoter in reversed orientation.

Tobacco leaves of transgenic plants fro· the greenhouse vere analysed for their GUS activity, depending on the various 5'promoter elements. The unit pmol MU/mg protein/min on the abscissa indicates enzyme activity. The ordinates respectively shov 3 out of 6-12 various tested plants. These plants reflect the highest, the average and the lowest activity of the respective tested plants, following vounding.

Germinating pollen of the construction pLSO34-571. The blue colouring in the pollen and in the pollen tube indicates the location of enzyme activity.

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MATERIAL AND METHODS

Reference is made to the following literature:

(1) Ansorge, V., De Mayer, L. (1980).

Thermally stabilized very thin (0.02-0.3 mm) polyacrylamide gels for electrophoresis. J. Chromatogr., 202: 45-53.

(2) Ansorge, V., 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, R., Breathnach, R., Chambon, P. (1980). The ovalbumin gene sequence of putative control regions. Nucl.

Acids Res., 8:127-142.

(5) Benton, V.D. and Davis, R.V. (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.Natl.Acad.Sci USA 84:

5745-5749.

- 10 Case 137-1046 (9) Fickett, F.tf. (1982).

Recognition of protein coding regions in DNA sequences. Nucl. Acids Res., 10:5303-5318.

(10) Frischauf, A.M., Lehrach, B., Poustka, A. and Murray, N. (1983). Lambda replacement vectors carrying polylinker sequences. J.

Mol. Biol., 170:827-842.

(11) Henikoff, S. (1984).

Unidirectional digestion vith exonuclease III creates targeted breakpoints for DNA sequencing. Gene, 28:351-359.

(12) Hoekema, A., Hirsch, P., Booykaas, 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., Vozney, J.M. and Boedtker, H. (1977). RNA molecular veight determinations by gele electrophoresis under denaturing conditions, a critical re-examination. Biochemistry, 16:4743.

(17) Lipphardt, S. (1988).

Dissertation an der Universitat zu Κδΐη.

APti0Ol81

Case 137-1046 (18) Logemann, J., Schell, J. and Villmitzer, 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, Nev York.

(20) Messing, J., Geraghty, 0., Ηυ, 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, Nev York: 211-227.

(21) Murashige, T., Skoog, F. (1962). A rapid method for rapid grovth and bloassays with tobacco tissue cultures. Physiol. Plant.,

15:473-497.

(22) Murray, M.G., Thompson, V.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 nev 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 vith 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

- 12 Case 137-1046 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 M13mp7derived system for insertion mutagenesis and sequencing vith synthetic universal primers. Gene, 19:259-268.

(28) Vassenegger, M. (1988).

Dissertation an der UniversitSt zu Κδΐη.

(29) Villmitzer, L., Schmalenbach, V. and Schell, J. (1981). Transcription of T-DNA in octopine and nopaline crovn gall tumours is inhibited by lov 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 vithout alteration of their normal capacity. EMBO J., 1:147-152.

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MATERIAL

Media

The media used for the culture of bacteria vere 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 % saccharose

3MSC : MS ♦ 3 X saccharose, 500 ug/ml claforan ·

MSC15 : US ♦ 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 vas added.

Strains and vectors

E. coli strains:

BMH 71-18: ώ (lac-proAB), thi, supE;

F'(lacl’, ZdeltaM15, proA*B⁺) (Messing et al. 1977)

C 600 : also knovn 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)

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Phages: EMBL4 (Frischauf et al., 1983)

M13mpl8 (Yanisch-Perron et al., 1985)

M13mpl9 ( )

Plants: Solanum tuberosum AM 80/5793 (haploid)

Nicotians tabacum Visconsin 38 (V38)

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., were carried out as described by Maniatis et al., (1982).

RNA Isolation

Isolation of RNA from various organs of the potato and of tobacco were carried out as described by Logemann et al., (1987).

Northern-Blot Analysis

The RNA was separated by electrophoresis on a 1.5X formaldehydeagarose-gel (Lehrach et al., (1977)). As described by Villmitzer et al., (1982), the RNA was subsequently transferred onto nitrocellulose, fixed and hybridized on ^3JP radioactively-labelled cDNA, washed and exposed.

DNA isolation

Nuclear DNA was recovered by the method of Bedbrook (1981) from potato leaves and used for cloning in lambda phages EMBL4. DNA from transformed tissue was purified by combined lyses with Triton X 100, SDS, and proteinase K (Vassenegger, 1988).

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Southern Blot Analysis

DNA vas separated by electrophoresis on 0.8 to 1.2X agarose gels, transferred onto nitrocellulose and fixed (Southern, 1975), also hybridized and washed as described by Villmitzer et al., (1981).

Establishment and research of a genomic bank

Isolation of genomic DNA

The genomic potato DNA used vas leaf DNA of the haploid species AM 80/5793, isolated by the method of Bedbrook, 1981. This DNA vas digested totally vith EcoRI.

Isolation of the phages OKA

EMBL4 DNA vas dissected vith EcoRI into three fragments, vhereby the tvo vector arms could be separated from the middle fragment by means of gel electrophoresis separation vith subsequent fragment isolation. In addition, commercially available, purified EMBL4 arms vere also used (Amersham).

Ligation and packaging

Following ligation of the EMBL4 arms vith the EcoRI-digested genomic DNA, the ligated, high molecular veight DNA is packaged in vitro into phage heads (Hohn and Murray, 1977; Hohn, 1979). The packaging material stems from a Lambda in vitro packaging kit from the company Amersham. The genomic bank vas plated out at a concentration of 25000 plaques per plate (25x25 cm).

Plaque hybridization

The discovery of cDNA homologous lambda clones vas made by means of plaque hybridization according to Benton and Davies (1977) using radioactively labelled cDNA. Positive hybridizing plaques vere

- 16 Case 137-1046 isolated and tested again.

DNA preparation of recombinant phages ml of a bacteria culture, lysed vith C600, are mixed vith 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 MgClj), and treated vith DNase and RNase. After extraction of the DNA by means of phenolization several times, the phage-DNA is EtOH-precipitated, vashed vith 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 vas cloned in M13mpl9 in both orientations. Since exonuclease III digests 5' projecting ends, but spares 3' projecting ends, 20 ug of the DNA to be analysed vas digested vith tvo restriction enzymes which produced a 5' and a 3' end on one side of the cloned fragment. Since the 5' end vas turned towards the fragment, exonuclease digestion into the fragment could take place. By successive stopping of this reaction, fragments which were 200 bp smaller could be isolated, and the projecting ends thereof were transformed into ligatable smooth ends by means of subsequent Sl-treatment. Finally, transformation of this DNA into BMH 7118 cells took place, vith subsequent single-stranded DNA isolation.

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Sequencing

Sequencing of single-stranded DNA was carried out by the chainterminating method of Sanger et al., (1977). Separation of the reaction products vas performed on 6X and 8X sequencing gels (Ansorge and de Mayer, 1980; Ansorge and Barker, 1984).

SI analysis

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Determination of the starting point of transcription vas effected according to Berk and Sharp (1987). For this purpose, the 1.2 kb fragment vas isolated from the plasmid pLSOOO vith EcoRI and Xhol, and dephosphorylated by treatment vith phosphatase. Then, radioactive labelling of the 5' OH ends took place through the combination of polynucleotide kinase and y- ³¹P-ATP. After denaturing of the DNA fragments, they vere hybridized vith 50 ug of whole RNA (hybridization buffer: 80X 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 oi hybridization starts at 80^eC and is lowered over night to 40°C. Subsequent Sl-nuclease digestion (120 U/ml) removes unpaired strands. It vas expected that the radioactively labelled Xhol cutting site found in the 5' untranslated region of the vunl gene would 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 vas transferred to A. tumefaciens LBA4404 (Hoekema et al., 1983) by the method described by Van Haute et al., (1983), by conjugation .

DNA analysis

Identification of DNA transfer to the Agrobacterium vas 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 provided evidence of the successful DNA transfer into the Agrobacterium.

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Transformation of tobacco

Culture of Agrobacterium

The Agrobacterium strains containing the appropriate LBA4404 vector required for infection vere grown in selective antibiotic medium (Zambrisky et al., 1983), sedimented by centrifugation and vashed in YEB medium without antibiotics. After further sedimentation and absorption in 3MS medium, the bacteria could be used for infection.

Infection of leaf sections

For infection of leaf sections, sterile leaves of tobacco species SNN and V38 vere used. Leaf pieces of about 1 cm^J size, immersed into the Agrobacterium suspension as described above, vith oo subsequent transfer onto 3MS medium. After incubation for 2 days at v 16 hours light and 25’C to 27°C, the pieces vere transferred onto MSC16 for callus and shoot induction. Shoots appearing after 4-6 weeks vere cut off and incubated on MSC15 medium.

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Analysis of the transformed plants

Isolation of genomic DNA

The DNA from the plants is isolated by a varied method of Murray and Thompson (1980), cut vith restriction enzymes and separated on a gel. After transfer of this DNA onto nitrocellulose, it is hybridized vith a radioactively labelled sample, vhich indicates the presence of this specific DNA in the genome of the plant under examination.

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Detection of GUS activity

Plants regenerated from transformation are examined using the method of Jefferson (1987) for the degree of activity of the enzyme 8-glucuronidase (GUS). An additional histochemical test (Jefferson, 1987) indicates localisation in the tissue. For this purpose, thin sections of the plants are incubated vith the substrate X-Gluc,

5-bromo-4-chloro-3-indolyl glucuronide. A blue precipitate forms at the site of enzyme activity.

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RESULTS

Establishment of a genomic bank from a haploid potato

The small number of vunl genes in the genome of the haploid potato cultivar AM8O/5793 led to the construction of a genomic bank consisting of genomic ONA which is digested with BcoRl.

ug of EcoRI cleaved DNA from AM80/5793 were ligated with EcoRI cleaved EMBL4 arms and plated on a C600 bacterial lawn. Ca. 500,000 plaques were obtained, which taken statistically, should represent the genome of the potato.

Identification of vunl homologous EMBL4 clones

After the transfer of these plaques onto nitrocellulose, by using plaque hybridization techniques on radioactively labelled wunlcONA, two genomic clones (wunl-22 and wunl-85) could be identified and purified.

Isolation of recombinant EMBL4 ONA from wunl-22, wunl-85, as well as their restriction mapping and hybridization on radioactive vunl- cDNA, gave the following data:

The vunl-cDNA equivalent fragment in vunl-85 has a size of 4 Kb. Thus, it corresponds exactly to the fragment size of BcoRl digested DNA from the haploid potato, which hybridizes with vunl-cDNA. Based on the asymmetrical Xhol cutting site in the 5'region of the cDNA clone, and the use of radioactive cDNA samples vhich 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 vunl 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 contained in vunl-85 could not be referred to for closer analysis of the vunl gene. Based on the digestion of the potato DNA with EcoRI, it is probable that during

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- 21 Case 137-1046 the subsequent ligation, tvo fragments not belonging together vere brought into the EMBL4 vector, that is, neither can they enter a functional relationship vith one another.

Sequence analysis of the genomic clone vunl-85

In the folloving, the genomic analysis of the vunl coding gene vas carried out. Since in the restriction behaviour, no differences cound be established between vunl-85 and vunl-22, the 4 Kb fragment of vunl-85 vas ligated into Eco RI cleaved pUC8 for further analysis (fig. 1). In order to sequence the vunl promoter and also the vunl gene, further re-cloning of the 4 Kb fragment into the Eco RI cutting site of M13mpl8 took place. Determination of its orientation vas carried out using control digestion at the asymmetrical Xhol cutting site. The clones 85*mpl8 and 85mpl8 represent both orientations of the fragment (fig. 62. Cutting of these plasmids vith SphI and Xbal enabled successive digestion, using exonucleaselll, of the 3' end of the vunl gene in the case of clone 85mpl8 and the 5' end of the vunl gene in the case of clone 85*mpl8 to take place.

Deletion clones resulting therefrom, vith varying fragment sizes, vere employed for sequencing. In all, it vas possible to sequence the entire vunl 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 vunl gene, the method of SI nuclease mapping vas 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 vunl gene vith vunl-mRNA, only the regions vhich because of their homology may form a double strand are protected from degradation of the single-strandspecific 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.

Case 137-1046

Assisted by the cutting sites Xho I and Eco RI, a 1.2 Kb fragment vas isolated from pLSOOO, and it contains the promoter and parts of the 5' untranslated region of the vunl gene. This fragment vas radioactively labelled vith ”P vith polynucleotide kinase at the 5' end of the Xho I site, and hybridized on 50 ug of total RNA from vounded potato tubers. After Si treatment of the hybrid, the size of the protected fragment vas determined on a sequencing gel; it varies betveen 162-179 bp (fig· 3)· When starting from the longest fragment, the start of transcription begins 179 bp upstream from the Xho I site, i.e. vith the sequence ACCATAC. This sequence conforms in the central region (CAT) vith 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, vieved from the start of transcription, a TATA box CTATATATT is found, vhich conforms veil vith the consensus sequence TCACTATATATAG determined by Joshi et al., 1987.

2. ) The CAAT box described by Benoist et al., 1980, in the region betveen -60 and -80 can be found in the vunl promoter at position -58 (CAAACT).

3. ) The 5' untranslated region of the vunl gene extends over 217 bp and is therefore comparatively large.

4. ) The vunl-mRNA coding gene thus encloses 794 bp, vhich corresponds very exactly vith the size determination of vunl-mRNA based on Northern-Blot analysis.

5. ) 97 bp of the 5' untranslated region in the vunl gene are missing in the cDNA clone vunl-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 vunl gene has no introns at its disposal.

I 8 1 0 0 0 dV

- 23 Establishment of the derivatives of the vector pFR69

The promoter deletion fragments produced by means of exonuclease III with the end points -571 bp, -111 bp and -86 bp, as well as the promoter up to the region -1022 bp in the 5' region, were recloned into vector pPR69 (Fig. 6). For preferential recloning, the deletion fragments beyond the Xhol cutting site were cut out of M13mpl8. To produce the control, which bears the vector in reversed Orientation, the 5' overlapping ends of the cutting sites were filled by the Klenov fragment of DNA-polymerase I and cloned into the vector. Absorption of the deletion fragments in the vector vas tested by hybridization on a radioactively labelled vunl promoter sample and by restriction analysis.

· #· -

Stable transformation and analysis of vunl-GUS in tobacco

Various vunl promoter deletions in transgenic plants vere tested for their organ specifity and their vound inducibility. For this purpose, the plasmids pLS034-1022, -571, -111, -86,-1O22R vere transformed into the agrobacterial strain LBA4404. These agrobacteria vere then used for transformation of the tobacco cultivar Wisconsin 38 (U38) by means of the leaf section infection method. Under kanamycin selection, the callus produced on the plant vere regenerated and vere examined on Southern Blot for correct integration of the pLS034-DNA into the plant genome.

The functional analysis of the transgenic tobacco plants vas extended to the detection of GUS activity in greenhouse plants.

Independent transformants of different constructions vere analysed for their GUS activity in various tissues. Non-trans formed tobacco plants served as a control (K).

Comparison vith the GUS activity in tobacco leaves shoved little activity for plants of the construction pLS034-86, -111 and

- 24 Case 137-1046 pLS034-1022R, vhich vas at about the level of activity of control plants. Compared vith these, the GUS activity of pLS034-571 is ca. 4 times higher, vhile the activity of the whole promoter is 13 to 370 times greater than that of the shortest construction (pLS034-86), see fig. 7.

The activity of the enzyme in various tissues of the plant vas analysed by means of histochemical in-situ analysis. For this, thin sections of the tissue vere incubated over night at 37®C vith 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 indicated that in plants of constructions pLS034-86, -111, -571 and -1022R, no activity can be detected in leaves, stem or roots.

Plants carrying the full vunl promoter in their construction show activity in all the said tissues.

Another picture vas seen during the analysis of flowers. In cross-sections of flowers of the various constructions, there vas the following distribution of enzyme activity: Plants of construction pLS034-1022 shoved GUS activity in the stoaiua of the anthers and in the pollen grain. 30X of the pollen grain shoved blue colouring after incubation vith X-Gluc. Anthers of pLS034-571 and -86 shoved the same pattern of activity, but vith less intensity (Pig. 8). In control plants, as vith plants of construction pLS034-lll and pLS034-1022R, no blue colouring could be detected in the stomium, and colouring of the pollen grain vas reduced to 0.75X.

Comparison of the measured GUS activity of anthers from the various constructions shoved 2-10 times stronger activity of pLS034-1022 than in pLSO34-571. The average enzyme activity of the full promoter in anthers reached 1276 pmol MU/mg/min. Anthers of the construction pLS034-571 shoved 2-times higher activity than anthers of pLS034-86. The average enzyme activity is 217 pmol MU/mg/min, resp. 97 pmol MU/mg/min.

AP 0 0 0 1 8 1

- 25 Case 137-1046

As is knovn, the 35S promoter of cauliflover 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 vhich is active in plants, the deletion fragments of the vunl promoter extend into this activity range throughout.

Claims (5)

1. CLAIMS: 1. DNA sequence having constitutive anther specific promoter activity, characterised in that it conprises the 5' upstream region of the wunl gene having a bp range of from -571 of the wunl promoter to +179 of the 5' untranslated region of the wunl gene, DNA sequences homologous thereto and parts thereof.
2. 2. DNA sequence according to Claim 1, with a bp range of

   a) from. -571 of the wunl promoter to +179 of the 5' untranslated region of the wunl gene, and DNA sequences homologous thereto, or

   b) from -571 of the wunl promoter to +1 of the 5' untranslated region, and DNA sequences homologous thereto, or

   c) from -86 of the wunl promoter to +179 of the 5' untranslated region, and w

   DNA sequences homologous thereto, or ®

   d) from -86 of the wunl promoter to +1 of the 5' untranslated region, and <

   DMA sequences homologous thereto, or <

   I

   e) from -571 to -111 of the wunl promoter 3- linked with the DNA sequence ' of from -86 of the wunl promoter to +179 of the 5- untranslated region, and DNA sequences homologous thereto, or

   f) from -571 to -111 of the wunl promoter 3' linked with the DNA sequence of from. -86 of the wunl promoter to +1 of the 'untranslated region, and DNA sequences homologous thereto.
3. 3. The use of a DNA sequence according to Claims 1 or 2 to obtain pollen/anther specific expression.
4. 4. The use according to Claim 3 for the preparation of plants having increased ornamental value or of male sterile plants.
5. 5. Transformation vectors comprising a constitutive anther-specific promoter

   -. ΑΛΙ?*' . ' · . ’ of Claims 1 or 2 operably linked to a structural gene element.

   BAD ORDINAL

   - 27 6. Plants or plant material containing a constitutive anther-specific promoter accor>

   iing to Claims 1 or 2.