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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)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• • 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

Definitions

• the present invention relates to a method of producing male sterile plants by use of a gene, which is expressible in plants and inhibits an essential cell function, hence disrupting full expression of a selected plant characteristic.
• Our International Patent Application No. WO 90/08831 describes and claims the disruption of respiration using a variety of disrupter genes which we refer to also as “pollen-inactivating genes”.
• An object of this invention is to provide genes for use in inhibiting gene expression.
• a method of inhibiting gene expression in a target plant tissue comprising stably transforming a plant cell of a type from which a whole plant may be regenerated with a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells ofthe target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting respiration in the cells ofthe said target tissue resulting in death ofthe cells characterised in that the said disrupter gene is selected from the group consisting ofthe T-urfl3 gene, genes encoding an ⁇ - or ⁇ -tubulin, short sense co-suppression of two essential maize cell cycle genes.
• ROA replication origin activator
• ANT adenine nucleotide transloc
• the ⁇ - or ⁇ -tubulin genes act as disrupters by de-stabilizing microtubule arrays in plant cells, hence inhibiting essential microtubule function in the said target tissue resulting in death ofthe cells.
• the promoter is an anther- and/or tapetum-specific promoter or a pollen-specific promoter, so that on expression ofthe said disrupter protein therein the regenerated plant is in male sterile. More preferably the said anther and/or tapetum-specific promoter was isolated using the cDNA sequences shown in Figure 1 or 2 or 3 ofthe accompanying drawings and using the techniques described in our International Patent Application No. WO 90/08826.
• Plasmid pMSlO in an Escherichia coli strain RR1 host containing the gene sequence shown in Figure 1 herewith, and deposited with the National Collection of Industrial & Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40090.
• promoters may also be used, for example a promoter such as the tapetum specific MFS 14 promoter.
• the present invention also provides a plant having stably incorporated in its genome by transformation a gene construct carrying a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells ofthe target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting an essential cell function such as respiration, microtubule arrays or cell division in the cells ofthe said target tissue resulting in death ofthe cells.
• the invention also provides a plant, particularly a monocotyledonous plant, and more particularly a corn plant, having stably incorporated within its genome a gene construct carrying a tissue-specific promoter which operates in the cells ofthe said target tissue and a disrupter gene encoding a protein which is capable of inhibiting an essential cell function such as respiration or microtubules in the said cells ofthe said target tissue resulting in death ofthe cells characterised in that the said disrupter gene is selected from the T-urfl3 gene, a short sense construct ofthe adenine nucleotide translocator, genes encoding an ⁇ - or ⁇ -tubulin and short sense down-regulation ofthe essential cell cycle genes, cdc25 and ROA.
• These gene constructs may be used as a means of inhibiting cell growth in a range of organisms from simple unicells to complex multicellular organisms such as plants and animals.
• tissue- or cell-specific promoters particular cells or tissue may be targeted and destroyed within complex organisms.
• One particular application intended for this invention is in the destruction of cells essential for male flower development, leading to male sterility.
• the invention therefore provides a method of preventing or inhibiting growth and development of plant cells based on gene constructs which inhibit an essential cell function such as respiration or microtubules.
• the technique has wide application in a number of crops where inhibition of particular cells or tissue is required. Of particular interest is the inhibition of male fertility in maize for the production of Fl hybrids in situ.
• tubulin genes are closely regulated by their endogenous promoters and closely matches the requirements of cells for these proteins which are polymerised and assembled into microtubules during growth and development ofthe plant.
• tubulin genes in an unregulated fashion using non-tubulin promoters in a particular tissue or stage of development, the equilibrium between free tubulin monomers and those polmerised in microtubules is disrupted resulting in instability ofthe microtubule complex and cellular dysfuntion.
• the tapetum or other cells ofthe anther this latter effect will cause the plants to be sterile.
• Transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation procedures are known from the literature such as agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, microprojectile transformation and pollen tube transformation, to mention but a few. Reference may be made to the literature for full details ofthe known methods.
• yeast The development and testing of these gene constructs as disrupters of mitochondrial function in the unicellular organism, yeast, will now be described. A mechanism by which these gene constructs may be used to inhibit plant cell growth and differentiation in transformed plants will also be described. The object of these procedures is to use yeast as a model system for the identification and optimisation of gene constructs for expressing proteins which disrupt mitochondrial function. Plant cells will then be transformed with the selected constructs and whole plants regenerated therefrom.
• Figure 1 shows the DNA sequence of an anther-specific cDNA, carried by plasmid pMSlO;
• Figure 2 shows the DNA sequence of a tapetum-specific cDNA, carried by plasmid pMS 14;
• Figure 3 shows the DNA sequence of an anther-specific cDNA, carried by plasmid pMS 18;
• Figure 4 shows the sequence ofthe T-urfl3 gene (SEQ ID NO 1) with the primers
• Turf-1 (SEQ ID NO 2) and Turf-2R (SEQ ID NO 3) underlined;
• Figure 5 shows DNA encoding the 59 amino acid region from the ATP-2 gene of Nicotinia phimbaginifolia ((SEQ ID NOS 4 and 5) with primers PREB-IB (SEQ ID NO 6) and PREB-R (SEQ ID NO 7) shown;
• Figure 6 shows the cleavage site ofthe pre- ⁇ sequence;
• Figure 7 is a map of vector pCaMVIiN
• Figure 8 is a map of vector RMS 17;
• Figure 9 is a map of vector pIE109;
• Figure 10 shows the MFS 14 promoter sequence (SEQ ID NO 8) with the following features: position 2198 transcription start CCT"A"CAA (concensus CTC'A' CA) position 2167 ATCC ATT (possible TATA box motif) position 2141 CCAT (possible CAAT box motif) position 2233 cdna start CAC"A"CAG position 2295 translation start GCAACAATGGCG (concensus TAAACAATGGCT);
• Figure 1 1 is a map of vector RMS 11;
• Figure 12 is a map of vector pMANT3
• Figure 13 illustrates the contruction of vectors for maize cell line transformation
• Figure 14 is a plot showing numbers of transformants produced in the various different experiments.
• PREB-R (5 ⁇ TCGGATCCCGCTGCGGAGGTAGCGTA-3', SEQ ID NO 7) designed using sequence information provided from Boutry et al (1987, Nature, 328. 341).
• the PCR conditions were as described above except that the annealing temperature was reduced to 60°C.
• PBEB-IB and PREB-2R were designed such that they introduce unique Kpnl and BamHI restriction sites at the 5' and 3' ends ofthe amplified fragment respectively. The position of these primers relative to the ATP2 gene is shown in Figure 5.
• the pre-B PCR fragment was digested with Kpnl and BamHI to generate cohesive ends and cloned into the corresponding sites ofthe vector pUC18 to give plasmid pPB 1.
• the TURF- 13 PCR product was then digested with BamHI and Xbal and cloned into the corresponding sites in pPB 1 to give plasmid pPB2.
• the pre- ⁇ sequence is fused in frame with the T-urfl3 gene so that following expression in a plant cell the full product will be transported to mitochondria. Cleavage ofthe pre- ⁇ sequence at the predicted site between residues 55-56 will release the T-urfl3 protein which includes at its NH 2 -terminus an additional 4 residues from the pre- ⁇ sequence ( Figure 6).
• the pre- ⁇ /T-urfl3 gene fusion in pBB2 removed by digestion with the enzymes Kpn I and Sal I, blunted-ended and cloned into the plasmid pCAMVIiN ( Figure 7) which was digested with BamHI and blunt-ended to give pPB3.
• This cloning step places the pre- ⁇ /T- urfl3 gene fusion under transcriptional control ofthe CAMV 35S promoter.
• the Adhl intron is present in this construct to boost expression levels in corn cells (Mascarenhas et al., 1990. Plant Mol. Biol., 15, 913-920) and the nos 3' sequence provides a polyA addition site.
• RMS 17 ( Figure 8) the PAT selection cassette from p IE 109 ( Figure 9) which allows in vitro selection of transformed corn cells on bialaphos was introduced as an EcoRI fragment into the unique EcoRI site of pPB3.
• the RMS 17 vector ( Figure 8) was introduced into cultured BMS cells using a silcon carbide fibre-mediated transformation technique as follows: Preparation of silicon carbide whiskers
• the Silar SC-9 whiskers were provided by the Advanced Composite Material Corporation Greer, South Carolina, USA.
• the sterile whisker suspensions were prepared in advance as follows. Approximately 50mg of whiskers were deposited into a pre-weighed 1.5 ml Eppendorf tube, which was capped and reweighed to determine the weight ofthe whiskers. The cap ofthe tube was perforated with a syringe needle and covered with a double layer of aluminium foil. The tube was autoclaved (121°C, 15psi, for 20 minutes) and dried.
• whisker suspensions were prepared for each experiment, as it had been reported that the level of DNA transformation when using fresh suspensions was higher than that of older suspensions.
• a 5% (weight/volume) whisker suspension was prepared using sterile deionised water. This was vortexed for a few seconds to suspend the whiskers immediately before use.
• Two positive control vectors were pPG3 which contains the PAT selection cassette alone and RMS 15, which is identical to RMS 17 except that the T-urfl 3 gene is replaced by the mitochondrial uncoupling protein gene, UCP, which jhas no effect on cultured BMS cells.
• the pre- ⁇ targeting sequence is present in both constructs.
• a negative control, which should completely prevent establishment of transgenic calli, was provided by RMS 13, which is identical to RMS 17 except that the pre ⁇ /T-urfl3 gene fusion is replaced by the cytotoxic ribonuclease gene, barnase.
• RMS11 RMS1 1 is a transformation vector in which expression ofthe pre- ⁇ /T-urf!3 gene fusion is controlled by the maize tapteum promoter, MFS 14.
• MFS 14 promoter and untranslated leader region from position -2198 to +97 is shown in Figure 10. In this way, expression ofthe T-urfl3 protein is limited to the cells producing pollen and not throughout the whole plant.
• RMS1 1 the Kpn I - Sail fragment from pPB2 containing the pre- ⁇ /T- urfl3 gene fusion was blunt-ended and ligated into the blunt ended BamHI site of plasmid pSC9 to yield pPB4.
• pPB4 the pre- ⁇ /T-urf!3 gene fusion is now positioned between the - 152 to +97 MFS 14 promoter fragment and the nos 3' polyadenylation sequence.
• This complete cassette was removed from pPB4 by digestion with Sacl and EcoRI and cloned into the corresponding sites of pSC7 to give plasmid pB5.
• pSC7 contains the -153 to -5800 region ofthe MFS 14 promoter so that the introduction ofthe Sacl - EcoRI fragment from pPB4 recreates the full 5.8 kb MFS 14 promoter.
• RMS 1 1 ( Figure 11) was completed by introduction ofthe PAT in vitro selection cassette from pl ⁇ 109 into the unique EcoRI site of pPB5.
• the maize transformation vector, RMS 1 1 was used to transform regenerable maize cell cultures by particle bombardment.
• Friable embryogenic Type II callus was initiated from immature zygotic embryos excised from either greenhouse or filed grown A188 plants 10-12 days after pollination with pollen from the inbred B73.
• the medium used for callus initiateion and maintenance was based onN6 medium as modified by Armstrong and Green. Specifically, the medium contained 6mM L-proline, 2% (w/v) sucrose, 2 mg/l 2,4-dichlorophenoxy- acetic acid (2,4-D) and 3% (w/v) Gelrite (Trade Mark, Caroline Biological Supply) at pH 6.0. Callus was grown for 4-4 weeks prior to suspension culture initiation.
• Suspension cultures were initiated in a MS-based liquid medium containing 100 mg/l myo-inositol, 2 mg/l 2,4-D, 2 mg/l 1-naphthaleneacetic acid (NAA), 6mM proline, 200 mg/l casein hydrolysate (Difco).
• MS-based liquid medium containing 100 mg/l myo-inositol, 2 mg/l 2,4-D, 2 mg/l 1-naphthaleneacetic acid (NAA), 6mM proline, 200 mg/l casein hydrolysate (Difco)
• each filter disc (with cells) was transferred to N6 based medium containing 100 mg/l myo-inositol, 2 mg l 2,4-D, 3% (w/v) sucrose, and 0.3% (w/v) Gelrite at pH 6.0.
• N6 based medium containing 100 mg/l myo-inositol, 2 mg l 2,4-D, 3% (w/v) sucrose, and 0.3% (w/v) Gelrite at pH 6.0.
• this medium was supplemented with
• 25 scoring 4 and below are functionally sterile.
• the accumulated sterility scores for each ofthe independent PCR positive clones is shown in Table 3 and compared to a maize line which was generated by bombardment with RMS1 1 but which is PCR negative for the MFS14/pre-B/T- urf!3 gene construct.
• Class 4 Slightly abnormal anthers with approximately 75 to 100 percent exertion.
• Class 4 through 5 tassels were considered fertile.
• the MANT-1 primer sequence appears at the beginning ofthe coding sequence of the gene and the MANT-2R primer sequence is near the end ofthe gene.
• the DNA was digested with Smal and subcloned into the Smal site of pUC18 to give pMANTl.
• the nos 3' polyadenylation signal sequence was introduced 3' to the ANT gene as a Sacl- EcoRI fragment into the corresponding sites in pMANTl to yield pMANT2.
• the objective of this experiment was to show that expression of pRMS-23 in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment of transgenic calli following transformation in two separate experiments.
• the vector DNAs were introduced into cultured BMS cells using the silcon carbide fibre transformation techniqiue as described in Example 2.
• the objective of this experiment was to show that expression of pTBR and pTBS in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment of transgenic calli following transformation.
• the vector DNAs were introduced into cultured BMS cells using the silcon carbide fibre transformation techniqiue as described in Example 2.
• MOLECULE TYPE DNA (genomic)
• TTCGGTTCTA TTTTTATTTT TTTTTTGTGC ATATTATTGA TAAAGGGATA TCTCCGTAAA 120 ATGGATGATT CCTATTTGGC TCAACTCTCC GAGTTAGCCA ACCACAATAG AGTGGAAGCG 180
• ORGANISM Turf-1 primer
• xi SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCT 36
• MOLECULE TYPE DNA (genomic)
• ORGANISM ATP-2 gene of Nicotinia plumbaginifolia
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• GGTTCTGTGT ATGTGTATCC GATATGTCCG GTGGTACATC TGAACTGGCC GGTTGTGTTA 540 GCTATTATTG GGGCGCCACG CGTAGCCCTG GTGCGGCCCG GACTATCCGG CAGAGAAAGC 600

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