WO1997038106A1 - Sequence d'un promoteur de gene extrait de la banane - Google Patents

Sequence d'un promoteur de gene extrait de la banane Download PDF

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Publication number
WO1997038106A1
WO1997038106A1 PCT/GB1997/000654 GB9700654W WO9738106A1 WO 1997038106 A1 WO1997038106 A1 WO 1997038106A1 GB 9700654 W GB9700654 W GB 9700654W WO 9738106 A1 WO9738106 A1 WO 9738106A1
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WIPO (PCT)
Prior art keywords
promoter
plant
banana
gene
acc oxidase
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PCT/GB1997/000654
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English (en)
Inventor
Gregory Dewitt May
Peter Barber Kipp
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Zeneca Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zeneca Limited filed Critical Zeneca Limited
Priority to AU19324/97A priority Critical patent/AU1932497A/en
Publication of WO1997038106A1 publication Critical patent/WO1997038106A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation

Definitions

  • the present invention relates to a gene promoter sequence isolated from banana and to the use of that promoter to regulate expression of chimeric genes in plants.
  • Gene expression is controlled by various regulatory components, including nucleic acid and protein elements.
  • gene expression is controlled by a region commonly referred to as the "promoter" which lies upstream (5') of the protein encoding region.
  • a promoter may be constitutive or tissue-specific, developmentally-regulated and/or inducible.
  • the core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence which defines the transcription start point for the structural gene.
  • the precise length of the core promoter region is indefinite but it is usually well -recognisable. Such a region is normally present, with some variation, in all promoters.
  • the base sequences lying between the various well-characterised “boxes” appear to be of lesser importance.
  • the presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemicals, hormones). Manipulation of crop plants to alter and/or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in plant tissues.
  • Such genetic manipulation therefore relies on the availability of means to drive and to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment.
  • Promoters and other regulatory components from bacteria, viruses, fungi and plants have been used to control gene expression in plant cells. Numerous plant transformation experiments using DNA constructs comprising various promoter sequences fused to various foreign genes (for example, bacterial marker genes) have led to the identification of useful promoter sequences. It has been demonstrated that sequences up to 500-1000 bases in most instances are sufficient to allow for the regulated expression of foreign genes. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants.
  • a range of naturally-occurring promoters are known to be operative in plants and have been used to drive the expression of heterologous (both foreign and endogenous) genes in plants: for example, the constitutive 35S cauliflower mosaic virus promoter, the ripening-enhanced tomato polygalacturonase promoter (Bird et al, 1988, Plant Molecular Biology, 1 1:651-662), the E8 promoter (Diekman & Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A1 1 promoter (Pear et al, 1989, Plant Molecular Biology,
  • ACC oxidase is an enzyme involved in the biosynthesis of ethylene.
  • abbreviation "ACC” means 1-aminocyclopropane-l-carboxylic acid.
  • Ethylene is a major plant hormone which has been shown to have a variety of effects on plant growth and development in many species. Endogenous levels of ethylene increase during several stages of development and in response to various stimuli including mechanical wounding and pathogen infection, ripening of climacteric fruits and leaf and flower senescence.
  • the biosynthetic pathway for ethylene in plants is well-established; for example, a review of ethylene biosynthesis was published by Yang and Hoffman in 1984 (Annual Review Plant Physiology, 35: 155-189). The final stages of ethylene biosynthesis proceed by the following pathway:
  • ACC cyclic amino acid 1-aminocyclopropane-l -carboxylic acid
  • EFE ethylene forming enzyme
  • ACC oxidase genes may be inco ⁇ orated into DNA constructs for plant transformation.
  • International patent application publication number WO91/01375 describes a method of modifying ethylene biosynthesis in plants by using DNA constructs based on genes encoding an enzyme involved in ethylene biosynthesis (such as ACC oxidase).
  • Sense constructs as well as antisense constructs may be used to regulate gene/enzyme activity.
  • An object of the present invention is to provide alternative promoters capable of driving gene expression in plants.
  • a DNA sequence encoding a banana ACC oxidase gene promoter capable of driving gene expression in plants having the sequence shown in SEQ ID NO 1 extending from nucleotide number 93 to 1448, or active variants thereof.
  • Active variants are DNA sequences partially homologous to SEQ ID NO1 or fragments thereof which retain promoter activity. It may be possible to alter the level or type of activity of the Banana ACC oxidase promoters by manipulating their sequences: for example, by altering the nucleotide sequence in key regulatory regions, by truncating the sequence or by deleting parts within the sequence.
  • Segments of the Banana ACC oxidase promoter sequences of between 100 and 2000 bases in length may be useful as plant-operative promoters.
  • the promoters of the invention are suitable for inco ⁇ oration into DNA constructs encoding any target gene so that the target gene is expressed when the construct is transformed into a plant.
  • the nucleotide sequence of the banana ACC oxidase promoter is shown as SEQ ID NO 1 : the ATG start codon is shown at the end of the promoter sequence (base number 1449 to 1451).
  • the putative TATA-box is between base number 1368 and base number 1373
  • Example 1 gives information on the limited homology between the banana ACC oxidase promoter and known promoters.
  • the banana ACC oxidase promoter may be synthesised ab initio using the sequence shown in SEQ ID NO 1 as a guide.
  • the promoter may be isolated from plant genomic DNA libraries using suitable probes derived from the said sequences or the promoter may be isolated using a PCR approach.
  • the promoter of the invention may be inserted as a promoter sequence in a recombinant gene construct destined for use in a plant. The construct is then inserted into the plant by transformation. Any plant species may be 5 transformed with the construct, and any suitable transformation method may be employed.
  • a plant gene expression cassette comprising the banana ACC oxidase promoter operatively linked 0 to a target gene, the promoter having the sequence shown as SEQ ID NO 1 or active variants thereof.
  • the target gene is a DNA sequence which may be derived from an endogenous plant gene or from a foreign gene of plant, fungal, algal, bacterial, viral 5 or animal origin. Normally it is a sequence other than the sequence encoding the
  • the target gene may be a single gene or a series of genes.
  • the target gene is adapted to be transcribed into functional RNA under the action of plant cell enzymes such as RNA polymerase.
  • RNA polymerase such as RNA polymerase.
  • o Functional RNA is RNA which affects the biochemistry of the cell: for example, it may be mRNA which is translated into protein by ribosomes or it may be RNA which inhibits the translation of mRNA related to it.
  • the target gene sequence may be a sense sequence encoding at least part of a functional protein or an antisense sequence. 5
  • the expression cassette is suitable for general use in plants.
  • the DNA construct comprising the expression cassette of the invention is inserted into a plant by transformation.
  • Any transformation method suitable for the target plant or plant cells may be employed, including infection by Agrobacterium tumefaciens o containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, microprojectile transformation, pollen tube transformation and transformation of plant cells using mineral fibres (US Patent Number 5302523, International Patent Application Publication Number WO94/28148).
  • the transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably inco ⁇ orated into the genome. Both transformed monocotyledonous and dicotyledonous plants may be obtained in this way.
  • Transgenic plant technology is for example described in the following publications: Swain WF, 1991, TLBTECH 9: 107-109; Ma JKC et_al, 1994, Eur J Immunology 24: 131-138: Hiatt A et al. 1992, FEBS Letters 307:71-75; Hein MB et , 1991, Biotechnology Progress 7: 455-461; Duering K, 1990, Plant Molecular Biology 15: 281-294.
  • genetically modified plants which may be produced include but are not limited to field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.
  • the invention further provides a plant cell containing a gene expression cassette according to the invention.
  • the gene expression cassette may be stably inco ⁇ orated in the plant's genome by transformation.
  • the invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom.
  • the invention further provides a method for controlling plant gene expression comprising transforming a plant cell with a plant gene expression cassette having an Banana ACC oxidase promoter operatively linked to a target gene, whereby the activated promoter drives expression of the target gene.
  • the promoter may be activated under certain spatial, temporal, developmental and/or environmental conditions.
  • the promoter fragments of the Banana ACC oxidase genes are fused to the GUS ( ⁇ -glucuronidase) reporter gene in DNA constructs suitable for plant transformation. GUS accumulation in transgenic plants may then be monitored.
  • GUS ⁇ -glucuronidase
  • clone 4 contained the entire ACO coding sequence (interrupted by 3 introns) as well as the associated 5' and 3' proximal regions.
  • the clone contained 1448 bases 5' of the predicted start codon (ATG) of the ACO coding sequence.
  • This putative promoter sequence (SEQ ID No 1) contains several regions of dyad symmetry and direct repeats.
  • Transformed banana plants containing the ACC oxidase promoter / GUS construct are produced by the method described in May et al (Biotechnology, 13, 486- 492, 1995). Transgenic plants are regenerated and grown to maturity. Fruit ripening enhanced expression of GUS is demonstrated by assay of enzyme activity in tissues at various stages of maturity.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne le promoteur du gène d'oxydase de l'acide 1-aminocyclopropane-1-carboxylique présent dans la banane, qui a la séquence nucléotidique SEQ-ID-NO-1 et qui est utilisé pour favoriser l'expression de gènes étrangers dans des plantes transgéniques.
PCT/GB1997/000654 1996-04-11 1997-03-11 Sequence d'un promoteur de gene extrait de la banane WO1997038106A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19324/97A AU1932497A (en) 1996-04-11 1997-03-11 Gene promoter sequence from banana

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9607700.3 1996-04-11
GBGB9607700.3A GB9607700D0 (en) 1996-04-11 1996-04-11 Gene promoter sequence from banana

Publications (1)

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WO1997038106A1 true WO1997038106A1 (fr) 1997-10-16

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GB (1) GB9607700D0 (fr)
WO (1) WO1997038106A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999015668A2 (fr) * 1997-09-25 1999-04-01 Boyce Thompson Institute For Plant Research, Inc. Proteines de banane, adn, et elements regulateurs d'adn associes au developpement du fruit
WO2000053763A1 (fr) * 1999-03-08 2000-09-14 Pioneer Hi-Bred International, Inc. Metallothioneine du mais et promoteur
WO2000056863A1 (fr) * 1999-03-19 2000-09-28 Agritope, Inc. Promoteurs de banane et de melon favorisant l'expression de transgenes dans des vegetaux
WO2011064224A1 (fr) * 2009-11-24 2011-06-03 Katholieke Universiteit Leuven, K.U.Leuven R&D Promoteurs de banane

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001375A1 (fr) * 1989-07-14 1991-02-07 Imperial Chemical Industries Plc Structures d'adn, cellules et plantes derivees de celles-ci

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001375A1 (fr) * 1989-07-14 1991-02-07 Imperial Chemical Industries Plc Structures d'adn, cellules et plantes derivees de celles-ci

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HUANG P. ET AL.: "Characterization and expression analysis of a banana gene encoding 1-aminocyclopropane-1-carboxylate oxidase", BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL, vol. 41, no. 5, 1997, pages 941 - 950, XP000675954 *
LOPEZ-GOMEZ R. ET AL.: "AC X91076", EMBL DATABASE, 11 September 1995 (1995-09-11), HEIDELBERG, XP002033394 *
LOPEZ-GOMEZ R. ET AL.: "Ethylene biosynthesis in banana fruit: Isolation of a genomic clone to ACC oxidase and expression studies", PLANT SCIENCE, vol. 123, no. 1-2, 1997, pages 123 - 131, XP000676021 *
ROSS G. ET AL.: "An ethylene-related cDNA from ripening apples", PLANT MOLECULAR BIOLOGY, vol. 19, no. 2, May 1992 (1992-05-01), pages 231 - 238, XP002033393 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999015668A2 (fr) * 1997-09-25 1999-04-01 Boyce Thompson Institute For Plant Research, Inc. Proteines de banane, adn, et elements regulateurs d'adn associes au developpement du fruit
WO1999015668A3 (fr) * 1997-09-25 1999-10-07 Thompson Boyce Plant Res Proteines de banane, adn, et elements regulateurs d'adn associes au developpement du fruit
US6284946B1 (en) 1997-09-25 2001-09-04 Boyce Thompson Institute For Plant Research Inc. Banana DNA associated with fruit development
WO2000053763A1 (fr) * 1999-03-08 2000-09-14 Pioneer Hi-Bred International, Inc. Metallothioneine du mais et promoteur
US6774282B1 (en) 1999-03-08 2004-08-10 Pioneer Hi-Bred International, Inc. Maize metallothionein gene and promoter
WO2000056863A1 (fr) * 1999-03-19 2000-09-28 Agritope, Inc. Promoteurs de banane et de melon favorisant l'expression de transgenes dans des vegetaux
US6642438B1 (en) 1999-03-19 2003-11-04 Exelixis Plant Sciences, Inc. Melon promoters for expression of transgenes in plants
AU782602B2 (en) * 1999-03-19 2005-08-11 Agrigenetics, Inc. Banana and melon promoters for expression of transgenes in plants
WO2011064224A1 (fr) * 2009-11-24 2011-06-03 Katholieke Universiteit Leuven, K.U.Leuven R&D Promoteurs de banane
US9057072B2 (en) 2009-11-24 2015-06-16 Katholieke Universiteit Leuven, K.U. Leuven R&D Banana promoters

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GB9607700D0 (en) 1996-06-19
AU1932497A (en) 1997-10-29

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