WO1997038106A1

WO1997038106A1 - Gene promoter sequence from banana

Info

Publication number: WO1997038106A1
Application number: PCT/GB1997/000654
Authority: WO
Inventors: Gregory Dewitt May; Peter Barber Kipp
Original assignee: Zeneca Limited
Priority date: 1996-04-11
Filing date: 1997-03-11
Publication date: 1997-10-16
Also published as: AU1932497A; GB9607700D0

Abstract

The promoter of the 1-aminocyclopropane-1-carboxylic acid oxidase gene in banana has the nucleotide sequence SEQ-ID-NO-1 and is used for driving expression of foreign genes in transgenic plants.

Description

GENE PROMOTER SEQUENCE FROM BANANA

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. In particular, 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.

Within the promoter region there are several domains which are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences, normally 70 base pairs immediately upstream of the gene. 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,

13:639-651) and many others.

As stated above, successful genetic manipulation relies on the availability of means to control plant gene expression as required. The scientist uses a suitable expression cassette (incorporating one or more promoters and other components) to regulate gene expression in the desired manner (for example, by enhancing or reducing expression in certain tissues or at certain developmental stages). The ability to choose a suitable promoter from a range of promoters having differing activity profiles is thus important.

In work leading to the present invention, we have isolated and fully sequenced an ACC oxidase gene promoter from banana. ACC oxidase is an enzyme involved in the biosynthesis of ethylene. In this document the 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:

Methionine ^■> S-adenosyl-L-methionine (SAM) -_►

1-aminocyclopropane-l -carboxylic acid (ACC) ^■* Ethylene.

The final step in the pathway of ethylene biosynthesis is the conversion of the cyclic amino acid 1-aminocyclopropane-l -carboxylic acid (ACC) to ethylene. This reaction is catalysed by the enzyme ACC oxidase (also known as "ethylene forming enzyme" or EFE) which was once thought to be constitutively expressed in most tissues. However, since the cloning of the gene the messenger RNA has been shown to be induced under a number of conditions known to result in increased ethylene production.

After the cloning of the first ACC oxidase cDNA clone from tomato, standard hybridisation procedures were used to isolate clones for ACC oxidase from other plant species. ACC oxidase cDNA or genomic clones have now been isolated from at least nine other species: ( 1 ) Melon (Cucumis melo)

Balague et al, 1993, Eur J Biochem, 212:27-34; (2) Petunia (Petunia hybrida)

Wang and Woodson, 1992, Plant Physiol, 100:535-536;

(3) Apple (Malus domestica)

Ross et al, 1992, Plant Molecular Biology, J9:231-238;

(4) Mustard (Brassica juncea) Pua et al, 1992, Plant Molecular Biology, J9;541-544;

(5) Avocado (Persea americana)

Christofferson et al, 1993, Cellular and molecular aspects of the plant hormone ethylene, Pech JC et al (eds), Kluwer, pages 65-71;

(6) Peach (Prunus persica) Callahan et al, 1992, Plant Physiol, 100:482-488:

(7) Orchid (Phalaenopsis)

Nadeau et al, 1993, Plant Physiol, 103:31-39:

(8) Kiwifruit (Actinidia deliciosa)

Macdiarmid and Gardiner, 1993, Plant Physiol, Ifll :691-692; (9) Carnation (Dianthus caryophyllus)

Wang et al, 1991, Plant Physiol, 96: 1000- 1001.

The whole or part of the protein coding regions of ACC oxidase genes may be incoφorated 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. According to the present invention, there is provided 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 incoφoration 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

The sequence of the banana ACC oxidase promoter has not previously been elucidated. 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. Alternatively, 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. In practice 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.

According to a second aspect of the invention, there is provided 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

ACC oxidase protein which follows the Banana ACC oxidase promoter in the naturally-occurring Banana ACC oxidase gene. 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. 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. Thus 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. In practice 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 incoφorated 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.

Examples of 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 incoφorated 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.

In order to determine their temporal and spatial expression, the promoter fragments of the Banana ACC oxidase genes are fused to the GUS (β-glucuronidase) reporter gene in DNA constructs suitable for plant transformation. GUS accumulation in transgenic plants may then be monitored. Example 3 describes some of these experiments.

The invention will now be described by way of example.

EXAMPLE 1

Isolation and characterisation of banana ACC oxidase promoter

A banana genomic DNA library prepared in the vector Lambda Fix II (Stratagene,

La Jolla, CA) was probed with the apple ACC oxidase cDNA clone Apy4 (Ross et. al. 1992, Plant Molecular Biology, 19: 231-238). Six clones containing sequences with homology to the apple ACC oxidase cDNA were identified by hybridisation. A 3365bp Sail fragment from one of the genomic clones - Banana ACC oxidase genomic clone 4 - was further characterised by sequence analysis.

Homology between clone 4 and the coding sequences of ACC oxidase genes from other species confirmed the identity of the genomic clone. 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.

EXAMPLE 2

Analysis of expression patterns of banana ACC oxidase gene.

Fruit ripening enhanced expression of ACC oxidase genes was demonstrated in banana pulp and peel. Total RNA was isolated from fruit pulp and peel samples of green bananas and 6 developmental stages after ripening had been induced by treatment with ethylene (>100μl/l for 24 hr). 10μg of RNA from each ripening stage and tissue were separated on formaldehyde denaturing gels and blotted onto nylon membrane. The membranes were pre- hybridised in 5x Denhardts, 5X SSC for 2hr at 65° and then hybridised overnight with random prime labelled banana ACO cDNA ACOS7 (Patent application No. ) at 65°C in fresh 5x Denhardts, 5X SSC. The membranes were then washed in 3x SSC,

0.1% SDS for 20 mins at 65°C and subseqently in 0.3x SSC, 0.1% SDS for 20 mins at 65°C prior to exposure to X-Ray film.

For both pulp and peel samples, there was very weak hybridisation to RNA from green unripe fruit. However, there was strong hybridisation of the ACO probe to pulp and peel RNA samples from all stages of ripening.

EXAMPLE 3

Banana ACC oxidase promoter - GUS constructs for analysis in transgenic plants

The 1386 bp Hindlll fragment (93 to 1479)fragment from the banana ACC oxidase genomic clone 4 was ligated into the Hindlll site of plant expression vector pBHOl (Clonetech, Palo Alto, CA, USA). This construction created a translational fusion between the banana ACC oxidase promoter and the E. coli β-glucuronidase (GUS) reporter gene:

1449-AGAGCGTGTC ATG GAT TCC TTT CCG GTT ATC GAC ATG GAG

Met Asp Ser Phe Pro Gly lie Asp Met Glu

Hindlll-AAG CTT GCA TGC CTG CAG GTC GAC TCT AGA GGA TCC Lys Leu Ala Cys Leu Gin Val Asp Ser Arg Pro Ser

CCG GGT GGT CAG TCC CTT ATG - Pro Gly Gly Gin Ser Leu Met - GUS

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.

SEQ ID No: 1 SEQUENCE TYPE: Nucleotide SEQUENCE LENGTH: 1484bp

STRANDEDNESS: Single

TOPOLOGY: Linear MOLECULE TYPE: DNA

ORIGINAL SOURCE ORGANISM: Banana (Musa acuminata). Cavendish IMMEDIATE EXPERIMENTAL SOURCE: Lambda ACC Oxidase clone 4

FEATURES:

From: 92 To: 97 HindlTI site

From: 1449 To: 1451 Putative start codon (ATG) From: 1479 To: 1484 Hindlll site

CGGCAAGTCA TGCATAGCTG CAAACATGTG ACAGGCACCG AACCAACAAT TGAAGAAGAT 60 ACGATAAACA TGCGTGAGCC TACATGCACC AAAGCTTGCC GACAAGTCAT GTTTGGGTGC 120 ACAATGTGTC CTCATCTTAC TTGCATATCT GCTGTTGCAC AACAGCAGAT TGCATGGAGG 180 TGTGTTTTCC GGCAATGCAA TCTTTGATGT TGGTTCTCTT NNCTCTCTTC TTGGATTGTT 240 TATAGCTCTG TTTCTTGTGC TCTTCTTTNA CGTAGATTCA TAGCGTAGCT TAAGTTGTTA 300 TAGATTACCT GTTTTACTGG GCAAACTTGT GCAACCCAGG AATATTCCCA TGTGCATCTT 360 CTTCCTGTTT CCTCTNTCAA ACTGTCTGTC ATGATGAGGN AGNACCGCTC TNGAGAATTC 420 CNATGTGGNT GGTTTACCCA TAACTGAGAG ATNTGTTGGC GTTCANTACA TGATGNTGTG 480 TCAGAANCAA TCTACCTGTT CTGGCCACGG AGGGTTTATG CGCAGTTCAA CGCCATTGGT 540 GGTGGTGTGG CTGCGTAATC CTTGGCTCCG TGCCCACGCA CATGACGAAC CCATTGTTTT 600 TTTGTGGCCA CCAACCGGAG AAGGGAGTCA AATAACTAGC GGACGGGAAT TTTCCCTTGA 660 CTTGTTCACT CACGTAAAGT GGTGAATTGG TAAAATTAAA CGGATCTATG GTCGGAGGAT 720 TAAGAAAAAC CCAGATAAGG GAGACCCATC CTTCACAAGT TGGACCTCGG CCGATTNGGC 780 CCGATCACCC TNTTTCACAC CGGATACTTA GTTTACGGCC ATTGCCGAAT GCCGACAACC 840 ATCGAGCGTT GTATTTAAGC AGGATGGCCC ATTTCTCAAA AACGAGAGGG ATACGAGTGG 900 AAAGGCCCTC TAATGAGCTG TGAACCGAAA CAATTTCTAC CCTATCGATC CCTGTTCTTT 960 TGATATGAAG TATACCCAAC AGTTCAAGAG AAGACGAGTA CACACGCATC GCCGATGCTG 1020 TGACGTTACT TTCTGAGTTG GCAATTTTGT CACTACAATC CAAGCGGAAG CCATGCACGC 1080 GAGGCGTCGC CATGGAAGAA CTCAACAACA TGATGCCTTC CCGGGTCTCC TCAAAGGGGA 1140 GAGACCGATG GAAGCAGCCA AACTTGGTCC CCGATCGTGA TGGGACGCGA GAGGTGGAAG 1200 CAAGGAGGGT GGAGAACCAG GCCAAAGGTG GTGGGGCTGA GAGATGGCCA ACTGGGTCAC 1260 CTTATGGAAT CGGCTCCGTT ACGTCTTCCA CTGCTGTTGC TCTCGTCGAT AGATCCTTCT 1320

CCAACTTTGC TTCTTCACTC ATTTCGTCCC TCGACGTCAA GAACGCCTAT AAATTGCCTG 1380

GTAATCAGCA GCACCTAGCA CACTCCAGAT AGAAAGCACA AGTGCAATCA GGGAAGAAAG 1440 AGCGTGTCAT GGATTCCTTT CCGGTTATCG ACATGGAGAA GCTT 1484

Claims

1. 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.

2. A DNA construct comprising a promoter as claimed in claim 1 operatively linked to a transcribable DNA region and a transcription termination signal.

3. A transgenic plant having stably incoφorated within its genome a DNA construct as claimed in claim 2.

4. A plant gene expression cassette comprising, in sequence, the banana ACC oxidase promoter claimed in claim 1, the coding region of a target gene and a 3' polyadenylation signal.