AU722034B2 - Genomic PPO clones - Google Patents

Description

WO 97/29193 PCT/AU97/00041 GENOMIC PPO CLONES The present invention relates to the isolation of genes encoding polyphenol oxidase (PPO) from plants.

Browning of plant tissues often occurs following. injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products.

Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.

It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed.

The prior art includes International Application PCT/AU92/00356 to the present applicant which describes the cloning of PPO genes from grapevine, broad bean leaf, apple fruit and potato tuber. This application recognises that PPO levels in plants may be manipulated by increasing or decreasing expression of PPO gene. The application also identifies two conserved copper binding sites in PPO genes, designated CuA and CuB, and predicts that these regions are suitable for design of probes and primers to obtain other plant PPO genes.

However, the method described in PCT/AU92/00356 suffers from the disadvantages that it is necessary to identify the appropriate stage of development of the target tissue, isolate mRNA or total RNA and synthesize cDNA. This in turn requires a relatively large amount of plant material.

Accordingly, it is an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art.

WO 97/29193 PCT/AU97/00041 In a first aspect of the present invention there is provided a method for preparing nucleic acid encoding PPO, fragments and derivatives thereof, which method includes providing a sample of plant tissue, a first primer in sense orientation having a sequence corresponding to a conserved region of a PPO gene, a second primer in antisense orientation having a sequence corresponding to a conserved region of a PPO gene; isolating genomic DNA from said plant tissue; and amplifying the genomic DNA using the first and second primers.

Surprisingly, the applicant has found that PPO genes in plants lack introns and, therefore, fragments of PPO genes may be amplified directly from genomic DNA of a range of plants. The lack of introns means that the size of the fragments can be predicted and bands of the appropriate size can be selected for cloning.

An advantage of the method of the present invention is that difficult steps of previous technologies (for example, identification of the appropriate stage of development of the target tissue, isolation of total and mRNA, first strand cDNA synthesis) are not required and genomic DNA encoding PPO or fragments thereof is readily isolated from a wide range of plants using relatively small amounts of tissue.

Since the sequences are derived from genomic DNA it is not possible to predict their temporal and spatial patterns of expression in the plant.

Nevertheless, if a clone expressed in a particular tissue, such as the fruit, is required such a clone may be identified by determining the expression pattern of each genomic clone using northern analysis. Alternatively, the genomic clones may be used to obtain cDNA clones from the target tissue.

Applicant has found that some of the genomic clones obtained using the method of the present invention have the same or a very similar nucleotide sequence to previously obtained cDNA clones which are described in, for example, International Patent Applications PCT/AU92/00356 and WO 97/29193 PCT/AU97/00041 PCT/AU96/00310 to the present applicant. This provides a validation of the method of the present invention and demonstrates that it can be used to obtain sequence of PPO genes which are known to be expressed in the target plants.

The terms "nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO" and "strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO gene" as used herein should be understood to refer to the strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO gene, respectively, or a sequence substantially homologous therewith, and may include presequences such as chloroplast transit sequence as well as sequences encoding mature PPO protein. For example, sequences which because of the degeneracy of the genetic code encode the same sequence of amino acids are encompassed by these terms.

The term "derivative" as used herein includes nucleic acids incorporating a catalytic cleavage site.

The plant tissue may be of any suitable type. Preferably, the plant tissue is leaf tissue, more preferably young leaf tissue. Preferably, the plant is strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat.

The genomic DNA may be isolated by any suitable method including extraction for example with a detergent such as CTAB. Methods for isolating genomic DNA from plants are well known to those skilled in the art and are described in, for example, Maniatis et al. "Molecular Cloning: A Laboratory Manual" Second Edition, Cold Spring Harbor Laboratory Press, 1989, the entire disclosure of which is incorporated herein by reference.

The first and second primers have sequences corresponding to conserved regions of PPO genes. These are identified by analysis of a number of known PPO genes.

The first primer may correspond to at least a portion of a first copper (Cu) binding site of PPO. Preferably, the first primer corresponds to at least a portion wn O7/21 o PCT/AU97/nnl41 4 of one of the CuA or CuB binding sites of PPO, which are described in International Application PCT/AU92/00356, the entire disclosure of which is incorporated herein by reference. The second primer may correspond to at least a portion of a second Cu binding site of PPO. Preferably, the second primer corresponds to at least a portion of the other of the CuA or CuB binding sites of

PPO.

The first and second primers may be degenerate. The first primer may include one or more of the following sequences or parts thereof: 5'-GCGAATTCTT[TC][TC]TICCITT[TC]CA[TC][AC]G-3' 5'-GCGAATTCGA[AG]GA[TC]ATGGGIAA[TC]TT[TC]TA-3' 5'-GCGAATTCAA[TC]GTIGA[TC][AC]GIATGTGG-3' 5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3' 5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3' 5'-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3' The second primer may include one or more of the following sequences or parts thereof: 5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3' 5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3' 5'-GCCTGCAGA[TC]A[GA]CTICCIGCAAACTC-3' 5'-GCCTGCAGTC[TC]TCIA[AG]IA[AG]ITCIG-3' The genomic DNA may be amplified using the polymerase chain reaction (PCR). Preferably the concentration of genomic DNA in the amplification mixture is approximately 0.4 to 4 ng/gl, more preferably approximately 1 to 3 ng/pl, most preferably approximately 2 ng/il.

Preferably the concentration of each of the primers in the amplification mixture is approximately 0.1 to 10 p.M, more preferably approximately 0.5 to 2 1 M, most preferably approximately 1 pM.

Preferably the amplification involves an initial denaturation at approximately 94°C for approximately 3 min, followed by approximately two cycles with denaturation at approximately 94 0 C for approximately 1 min, annealing at approximately 370C for approximately 2 min, a slow ramp to approximately 720C over approximately 2 min and elongation at approximately Wd-1 O"7/01 al TTQ'7/nnA 1 72°C for approximately 3 min, followed by approximately 25 cycles of denaturation at approximately 941C for approximately 1 min, annealing at approximately 55 0 C for approximately 1 min, and elongation at approximately 72 0 C for approximately 3 min.

Those skilled in the art will appreciate that if the conserved regions are internal, the nucleic acid isolated will be a fragment of the PPO gene lacking 3' and 5' termini. However, using methods known to those skilled in the art, including the methods described in International Patent Application PCT/AU96/00310 to the present applicant, the entire disclosure of which is incorporated herein by reference, it is possible to determine the complete nucleic acid sequence of the PPO gene and to prepare or isolate nucleic acid encoding such PPO or antisense to such PPO.

In a further aspect of the present invention, there is provided a nucleic acid encoding strawberry PPO or antisense to strawberry PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 1 or 2 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 3 or 4 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding apricot PPO or antisense to apricot PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 5 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding avocado PPO or antisense to avocado PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 6 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding cherry PPO or antisense to cherry PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 7 or 8 or an homologous sequence.

I\Mt" 01710 t P'T/ATTQO/nnA 1 In a further aspect of the present invention, there is provided a nucleic acid encoding peach PPO or antisense to peach PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 9 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding pear PPO or antisense to pear PPO, fragments and derivatives thereof.

Preferably the nucleic acid has the sequence shown in Figure 10 or 11 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof.

In a further aspect of the present invention, there is provided a nucleic acid encoding tea PPO or antisense to tea PPO, fragments and derivatives thereof.

In a further aspect of the present invention, there is provided a nucleic acid encoding coffee PPO or antisense to coffee PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 12 or 13 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding apple PPO or antisense to apple PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 14 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding lettuce PPO or antisense to lettuce PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 15 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding French bean PPO or antisense to French bean PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 16, 17 or 18 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 19 or WO 97/29193 PCT/AU97/00041 7 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding rice PPO or antisense to rice PPO, fragments and derivatives thereof.

Preferably the nucleic acid has the sequence shown in Figure 21 or 22 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding potato PPO or antisense to potato PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figure 23, 24, or 26 or an homologous sequence.

In a further aspect of the present invention, there is provided a nucleic acid encoding parsnip PPO or antisense to parsnip PPO, fragments and derivatives thereof.

In a further aspect of the present invention, there is provided a nucleic acid encoding wheat PPO or antisense to wheat PPO, fragments and derivatives thereof.

The nucleic acid may be provided in an isolated and/or purified form.

Fragments of the nucleic acid sequence are preferably functionally active ie. they encode a polypeptide that possesses the relevant PPO activity or they encode a relevant epitope.

The nucleic acid may be prepared by a method as hereinbefore described.

The nucleic acid may be modified, for example by inclusion of a catalytic cleavage site.

In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or antisense to such PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or antisense to such PPO, fragments and derivatives thereof; and WO 97/29193 PCT/AU97/00041 8 a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.

The nucleic acid may be prepared by a method as hereinbefore described.

The nucleic acid may be modified, for example by inclusion of a catalytic cleavage site.

The vector may be a plasmid expression vector. For example Bluescript SK+ has been found to be suitable. Alternatively, the vector may be a binary vector. The recombinant vector may contain a promoter, preferably a constitutive promoter upstream of the nucleic acid.

The cloning step may take any suitable form. A preferred form may include fractionating the cDNA, for example on a column or a gel; isolating a fragment of the expected size, for example from the column or gel; and ligating said fragment into a suitable restriction enzyme site, for example the EcoRV site of a Bluescript SK' vector.

In order to test the clones so formed, a suitable microorganism may be transformed with the vector, for example by electroporation, the microorganism cultured and the polypeptide encoded therein expressed. The microorganism may be a strain of Escherichia coli, for example E.coli DH5 has been found to be suitable. Alternatively, appropriate vectors may be used to transform plants.

-In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or antisense to such PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.

The nucleic acid may be prepared by a method as hereinbefore described.

The nucleic acid may be modified, for example by inclusion of a catalytic cleavage site.

The vector may be a plasmid expression vector. For example Bluescript WO 97/29193 PCT/AU97/00041 9 SK has been found to be suitable. Alternatively, the vector may be a binary vector. The recombinant vector may contain a promoter, preferably a constitutive promoter upstream of the nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or antisense to such PPO, fragments and derivatives thereof.

The microorganism may be a strain of Escherichia coli, for example E.coli has been found to be suitable.

In a further aspect of the present invention there is provided a method of decreasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO, a modified nucleic acid encoding such PPO, or a nucleic acid antisense to strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO, fragments and derivatives thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

PPO activity may be decreased by the use of sense constructs (cosuppression). Alternatively the nucleic acid may include a sequence encoding antisense mRNA to strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or a fragment thereof. Alternatively the nucleic acid may encode strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or fragment thereof and incorporate a catalytic cleavage site (ribozyme). The nucleic acid may be included in a recombinant vector as hereinbefore described.

In a preferred aspect, the nucleic acid may be included in a binary vector. In a further preferred aspect, the introduction of a binary vector into the plant may be WO 97/29193 PCT/AU97/00041 by infection of the plant with an Agrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles. Methods for transforming plants with Agrobacterium are known to those skilled in the art and are described in, for example, May et al., Bio/technology (1995) 13:486-492, the entire disclosure of which is incorporated herein by reference. Methods for transforming plants by bombardment with DNA coated microprojectiles are known to those skilled in the art and are described in, for example, Sagi et al., Bio/technology (1995) 13:481-485, the entire disclosure of which is incorporated herein by reference.

In a further aspect of the present invention there is provided a method of increasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

The nucleic acid may be included in a recombinant vector as hereinbefore described. In a preferred aspect, the nucleic acid may be included in a binary vector. In a further preferred aspect, the introduction of the binary vector into the plant may be by infection of the plant with an Agrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles.

The plant may be of any suitable type. However the method is particularly applicable to strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat, respectively.

In a further aspect of the present invention there is provided a transgenic plant, which plant contains nucleic acid capable of modifying expression of the normal PPO gene.

The plant may be of any suitable type. Preferably, the plant is strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, WO 97/29193 PCT/AU97/00041 11 lettuce, French bean, banana, rice, potato, parsnip or wheat.

The nucleic acid may be as hereinbefore described.

In a still further aspect of the present invention there is provided a plant vaccine including nucleic acid encoding strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip or wheat PPO or antisense to such PPO, fragments and derivatives thereof.

The present invention will now be more fully described with reference to the accompanying Example. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the Figures: FIGURE 1: The genomic nucleotide sequence and derived protein sequence GSPO2 encoding PPO from strawberry.

FIGURE 2: The genomic nucleotide sequence and derived protein sequence GSPO6 encoding PPO from strawberry.

FIGURE 3: The genomic nucleotide sequence and derived protein sequence GTPO1 encoding PPO from tobacco.

FIGURE 4: The genomic nucleotide sequence and derived protein sequence GTP03 encoding PPO from tobacco.

FIGURE 5: The genomic nucleotide sequence and derived protein sequence GAPO3 encoding PPO from apricot.

FIGURE 6: The genomic nucleotide sequence and derived protein sequence GAVPO1 encoding PPO from avocado.

FIGURE 7: The genomic nucleotide sequence and derived protein sequence GCPO1 encoding PPO from cherry.

FIGURE 8: The genomic nucleotide sequence and derived protein sequence GCPO2 encoding PPO from cherry.

FIGURE 9: The genomic nucleotide sequence and derived protein sequence GPCPO2 encoding PPO from peach.

FIGURE 10: The genomic nucleotide sequence and derived protein sequence GPEPPO1 encoding PPO from pear.

WO 97/29193 FIGURE 11: The genomic nucleotide sequence sequence GPEPPO2 encoding PPO from pear.

FIGURE 12: The genomic nucleotide sequence sequence GCOPO3 encoding PPO from coffee.

FIGURE 13: The genomic nucleotide sequence sequence GCOPO4 encoding PPO from coffee.

FIGURE 14: The genomic nucleotide sequence sequence GALPO3 encoding PPO from apple.

FIGURE 15: The genomic nucleotide sequence sequence GLEPO1 encoding PPO from lettuce.

FIGURE 16: The genomic nucleotide sequence sequence GFPO2 encoding PPO from French bean.

FIGURE 17: The genomic nucleotide sequence sequence GFPO3 encoding PPO from French bean.

FIGURE 18: The genomic nucleotide sequence sequence GFPO4 encoding PPO from French bean.

PCT/AU97/00041 and derived protein and derived protein and derived protein and derived protein and derived protein and derived protein and derived protein and derived protein and derived protein FIGURE 19: The genomic nucleotide sequence GBPO2 encoding PPO from banana.

FIGURE 20: The genomic nucleotide sequence GBPO6 encoding PPO from banana.

FIGURE 21: The genomic nucleotide sequence GRPO5 encoding PPO from rice.

FIGURE 22: The genomic nucleotide sequence GRPO6 encoding PPO from rice.

FIGURE 23: The genomic nucleotide sequence GPOT2 encoding PPO from potato.

FIGURE 24: The genomic nucleotide sequence GPOT6 encoding PPO from potato.

FIGURE 25: The genomic nucleotide sequence GPOT8 encoding PPO from potato.

FIGURE 26: The genomic nucleotide sequence GPOT10 encoding PPO from potato.

sequence sequence and sequence and sequence and sequence and sequence and sequence and sequence and derived protein derived protein derived protein derived protein derived protein derived protein derived protein WO 97/29193 PCT/AU97/00041 13 EXAMPLE 1 Isolating Genomic PPO Clones Genomic DNA was isolated from young leaf tissue of a range of different plants using the hot CTAB method described by Stewart, CN and Via, LE (1993) "A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications" BioTechniques 14:748-750, the entire disclosure of which is incorporated herein by reference. Based on known plant PPO DNA sequences, oligonucleotide primers were designed in the conserved regions of the gene, particularly in the regions which encode the two copper binding sites, CuA and CuB (Dry, IB and Robinson, SP (1994) "Molecular cloning and characterisation of grape berry polyphenol oxidase" Plant Molecular Biology 26:495-502; Thygesen, PW, Dry, IB and Robinson, SP (1995) "Polyphenol oxidase in potato. A multigene family that exhibits differential expression patterns" Plant physiology 109:525-531, the entire disclosures of which are incorporated herein by reference). A number of forward primers (GEN3-4 and GEN7-10) and reverse primers (REV1-4) were synthesised: GEN3: (5'-GCGAATTCTT[TC][TC]TICCITT[TC]CA[TC][AC]G-3') GEN4: (5'-GCGAATTCGA[AG]GA[TCATGGGIAA[TC]TT[TC]TA-3') GEN7: (5'-GCGAATTCAA[TC]GTIGA[TC][AC]GIATGTGG-3') GEN8: (5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3') GEN9 (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') (5'-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3') REV1 (5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3') REV2: (5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3') REV3: (5'-GCCTGCAGA[TC]A[GA]CTICCIGCAAACTC-3') REV4: (5'-GCCTGCAGTC[TC]TCIA[AG]IA[AG]ITCIG-3') The genomic DNA (50ng) was amplified by PCR in a 25dl reaction using combinations of the GEN and REV primers (see Table each at a final concentration of 1 tM.

WO 97/29193 PCT/AU97/00041 14 Table 1I PPO Genomnic Clones Plant Clone# Fig# Size 5'-Primer Comments Primer Strawberry GSP02 1 590 GEN3 REV2 Strawberry GSP04 ___440 GEN8 REVI 76% same as GSP02 Strawberry GSP05 ___527 GEN8 REV2 96% same as GSP02 Strawberry GSP06 2 527 GEN8 REV2 98% same as GSP04 Tobacco GTPO1 3 465 GEN8 REVi Tobacco GTP02 ___459 GEN8 REVi 97% same as GTP03 Tobacco GTP03 4 545 GEN8 REV2 69% same as GTPOI Apricot GAP01 465 GEN8 REWi Apricot GAP02 ___465 GEN8 -REVI same as GAP01 Apricot GAP03 5 548 GEN8 REV2 same as GAP01 Avocado GAVP01 6 590 GEN3 REV2 Avocado GAVP02 ___590 GEN3 REV2 95% same as GAVP01 Cherry GCP01 7 590 GEN3 REV2 Cherry GCP02 8 442 GEN8 REVI 67% same as GCPO01 Peach GPCP01 ___465 GEN8 REVI Peach GPCP02 9 548 GEN8 REV2 98% same as GPCP01 Pear GPEPPO1 10 465 GEN8 REWi Pear GPEPP02 11 527 GEN8 REV2 56% same as GPEPPOI Pear GPEPP03 ___548 GEN8 REV2 same as GPEPPO1 Coffee GCOP03 12 674 GEN9 REV2 Coffee GCOP04 13 667 GEN9 REV2 94% same as GCOP03 Apple GALP03 14 588 GEN9 REWi Lettuce GLEP01 15 671 GEN9 REV2 70% same as LP01 French bean GFP02 16 668 GEN9 REV2 French bean GFP03 17 689 GEN9 REV2 63% same as GFP02 French bean GFP04 18 662 GEN9 REV2 65% same as GFP02 Banana GBPO2 19 426 GEN10 REVi =BANPPOI (cDNA) Banana GBPO6 20 512 GEN10 REV2 53% same as GBPO2 Rice GRP05 21 662 GEN10 REV2 (contains an intron9 Rice jIJ~ GRPO 22 62 GN1O REV2 6j4% same as Potato GPOT2 23 -551 -GEN8 REV2 75% same as P0T33 Potato GPOT6 24 682 GEN9 REV2 Same as P0T32 (cDNA) Potato GPOT8 25 -536 GEN10 REV2 71% same as P0T33 Potato GPOT10 26 -515 -GEN10 REV2 97% same as P1&P2 (cDNA) Amplification involved an initial denaturation at 940 C for 3 min, followed by two cycles with denaturation at 940 C for 1 min, annealing at 370 C for 2 min, a slow ramp to 720 C over 2 min and elongation at 720 C for 3 min, followed by cycles of denaturation at 940 C for 1 min, annealing at 550 C for 1 min, and elongation at 720 C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidiumn bromide to determine the size of the PCR 117/ 11 019 P"T/AI ATT"mnnA products. Where bands of the predicted size were identified, the remainder of the DNA was purified and concentrated using PCR Wizard Prep columns (Promega Corporation) or a QIAquick Spin PCR Purification Kit (QIAGEN Inc).

The purified DNA was electrophoresed on a 2% Nusieve agarose gel and bands of the appropriate size were excised and ligated into Eco RV-cut Bluescript SK vector (Stratagene) which had been T-tailed with Taq Polymerase. The ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Identity of PPO clones was established by homology with known plant PPO gene sequences.

Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims (79)

1. A method for preparing nucleic acid encoding PPO, fragments and derivatives thereof, which method includes: providing a sample of plant tissue, a first primer in sense orientation having a sequence corresponding to a conserved region of a PPO gene, a second primer in antisense orientation having a sequence corresponding to a conserved region of a PPO gene; isolating genomic DNA from said plant tissue; and amplifying the genomic DNA using the first and second primers, wherein said conserved region of a PPO gene corresponds to or is complementary to a nucleotide sequence encoding a copper (Cu) binding site of PPO.

2. The method according to claim 1 wherein the sequence of the fist primer corresponds to a portion of or in close proximity to a nucleotide sequence encoding a first copper (Cu) o binding site of PPO.

3. The method according to claim 2 wherein the first copper (Cu) binding site is one of CuA or CuB binding sites of PPO.

4. The method according to claim 1 wherein the first primer includes one or more of the sequence or parts thereof selected from the group consisting of: 5'-GCGAATTCTT[TC][TC]TICCITT[TC]CA[TC][AC]G-3' 9 °5'-GCGAATTCGA[AG]GA[TC]ATGGGIAA[TC]TT[TC]TA-3' 5'-GCGAATTCAA[TC]GTIGA[TC][AC]GIATGTGG-3' 5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3' 5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3' 5'-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3' P:\OPER\MRO\14330-97.CLM 1/2/00 -17- The method according to claim 1 wherein the sequence of the second primer corresponds to a portion of or in close proximity to a nucleotide sequence encoding a second copper (Cu) binding site of PPO.

6. The method according to claim 5 wherein the second copper (Cu) binding site is one of CuA or CuB binding sites of PPO.

7. The method according to claim 1 wherein the second primer includes one or more of the sequences or parts thereof selected from the group consisting of: 5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3' 5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3' 5'-GCCTGCAGA[TC]A[GA]CTICCIGCAAACTC-3' 5'-GCCTGCAGTC[TC]TCIA[AG]IA[AG]ITCIG-3'

8. The method according to claim 1 wherein the plant tissue is derived from a plant selected from the group consisting of strawberry, tobacco, apricot, avocado, cherry, peach, pear, coffee, apple, lettuce, French bean, banana, rice or potato. 3

9. The method according to claim 1 wherein the genomic DNA is ampliied using the polymerase chain reaction (PC) and the genomic DNA used in the PCR is approximately 0.4 to 4ng/jpl.

10. The method according to claim 9 wherein the concentration of each of the first and S. second primers is approximately 0.1 to 10 M. i. :11. An isolated nucleic acid prepared by the method according to claim 1. a

12. The nucleic acid according to claim 11 further including a catalytic cleavage site. S13. An isolated nucleic acid comprising a nucleotide sequence which encodes strawberry 1- PPO or a fragment or derivative thereof. P:\OPER\MRO\14330-97.CLM 1/2/00 -18-

14. The isolated nucleic acid according to claim 13 wherein the nucleotide sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure 1; (ii) the nucleotide sequence set forth in Figure 2; (iii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 1; and (iv) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 2. The isolated nucleic acid according to claim 13 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

16. An isolated nucleic acid comprising a nucleotide sequence which encodes tobacco PPO or a fragment or derivative thereof.

17. The isolated nucleic acid according to claim 16 wherein the nucleotide sequence is oselected from the group consisting of: the nucleotide sequence set forth in Figure 3; (ii) the nucleotide sequence set forth in Figure 4; (iii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 3; and (iv) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 0 4. S. S <i

18. The isolated nucleic acid according to claim 16 wherein the fragment comprises a °nucleotide sequence encoding one or more copper (Cu) binding sites of PPO. s

19. An isolated nucleic acid comprising a nucleotide sequence which encodes apricot PPO or a fragment or derivative thereof. The isolated nucleic acid according to claim 19 wherein the nucleotide sequence is P:\OPER\MRO\14330-97.CLM 1/2/00 -19- selected from the group consisting of: the nucleotide sequence set forth in Figure (ii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure

21. The isolated nucleic acid according to claim 19 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

22. An isolated nucleic acid comprising a nucleotide sequence which encodes avocado PPO or a fragment or derivative thereof.

23. The isolated nucleic acid according to claim 21 wherein the nucleotide sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure 6; (ii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 6

24. The isolated nucleic acid according to claim 21 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

25. An isolated nucleic acid comprising a nucleotide sequence which encodes cherry PPO or a fragment or derivative thereof.

26. The isolated nucleic acid according to claim 25 wherein the nucleotide sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure 7; (ii) the nucleotide sequence set forth in Figure 8; (iii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 7; and RA* (iv) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 8. P:\OPER\MRO\14330-97.CLM 1/2/00

27. The isolated nucleic acid according to claim 25 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

28. An isolated nucleic acid comprising a nucleotide sequence which encodes peach PPO or a fragment or derivative thereof.

29. The isolated nucleic acid according to claim 28 wherein the nucleotide sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure 9; (ii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 9. The isolated nucleic acid according to claim 28 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

31. An isolated nucleic acid comprising a nucleotide sequence which encodes pear PPO or a fragment or derivative thereof.

32. The isolated nucleic acid according to claim 31 whereiin the nucleotid sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure (ii) the nucleotide sequence set forth in Figure 11; (iii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 10; and (iv) a nucleotide sequence that encodes the amino acid sequence set forth in Figure S 11.

33. The isolated nucleic acid according to claim 31 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO. An isolated nucleic acid comprising a nucleotide sequence which encodes coffee PPO P:\OPER\MRO\14330-97.CLM 1/2/00 -21- or a fragment or derivative thereof. The isolated nucleic acid according to claim 34 wherein the nucleotide sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure 12; (ii) the nucleotide sequence set forth in Figure 13; (iii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 12; and (iv) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 13.

36. The isolated nucleic acid according to claim 34 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

37. An isolated nucleic acid comprising a nucleotide sequence which encodes apple PPO or a fragment or derivative thereof when isolated by a process comprising the method according to any one of claims 1 to

38. The isolated nucleic acid according to claim 37 wherein the nucleotide sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure 14; S' (ii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 14. e* S

39. The isolated nucleic acid according to claim 37 wherein the fragment comprises a S nucleotide sequence encoding one or more copper (Cu) binding sites of PPO. An isolated nucleic acid comprising a nucleotide sequence which encodes lettuce PPO or a fragment or derivative thereof. The isolated nucleic acid according to claim 40 wherein the nucleotide sequence is P:\OPER\MRO\14330-97.CLM 1/2/00 -22- selected from the group consisting of: the nucleotide sequence set forth in Figure (ii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure

42. The isolated nucleic acid according to claim 40 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

43. An isolated nucleic acid comprising a nucleotide sequence which encodes French bean PPO or a fragment or derivative thereof when isolated by a process comprising the method according to any one of claims 1 to

44. The isolated nucleic acid according to claim 43 wherein the nucleotide sequence is selected from the group consisting of: the nucleotide sequence set forth in Figure 16; (ii) the nucleotide sequence set forth in Figure 17; (iii) the nucleotide sequence set forth in Figure 18; (iv) the nucleotide sequence that encodes the amino acid sequence set forth in Figure 16; a nucleotide sequence that encodes the amino acid sequence set forth in Figure 17; and (vi) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 18.

45. The isolated nucleic acid according to claim 43 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

46. An isolated nucleic acid comprising a nucleotide sequence which encodes banana PPO or a fragment or derivative thereof. The isolated nucleic acid according to claim 46 wherein the nucleotide sequence is P:\OPER\MRO\14330-97.CLM 1/2/00 -23- selected from the group consisting of: the nucleotide sequence set forth in Figure 19; (ii) the nucleotide sequence set forth in Figure (iii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 19; and (iv) a nucleotide sequence that encodes the amino acid sequence set forth in Figure

48. The isolated nucleic acid according to claim 46 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

49. An isolated nucleic acid comprising a nucleotide sequence which encodes rice PPO or a fragment or derivative thereof. The isolated nucleic acid according to claim 49 wherein the nucleotide sequence is 0 selected from the group consisting of: the nucleotide sequence set forth in Figure 21; S the nucleotide sequence set forth in Figure 22; o~oq r_ 1_ (iii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 21; and (iv) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 22.

51. The isolated nucleic acid according to claim 49 wherein the fragment comprises a nucleotide sequence encoding one or more copper (Cu) binding sites of PPO.

52. An isolated nucleic acid comprising a nucleotide sequence which encodes potato PPO or a fragment or derivative thereof when isolated by a process comprising the method according to any one of claims 1 to The isolated nucleic acid according to claim 52 wherein the nucleotide sequence is P:\OPER\MRO\14330-97.CLM 1/2/00 -24- selected from the group consisting of: a nucleotide sequence set forth in Figure 23; (ii) a nucleotide sequence set forth in Figure 24; (iii) the nucleotide sequence set forth in Figure (iv) the nucleotide sequence set forth in Figure 26; a nucleotide sequence that encodes the amino acid sequence set forth in Figure 23; (vi) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 24; (vii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure and (viii) a nucleotide sequence that encodes the amino acid sequence set forth in Figure 26.

54. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 13 to

55. An antisense molecule comprising the isolated nucleic acid of claim 54.

56. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 16 to 18.

57. An antisense molecule comprising the isolated nucleic acid of claim 56.

58. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 19 to 21.

59. An antisense molecule comprising the isolated nucleic acid of claim 58. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 22 to 24. P:\OPER\MRO\14330-97.CLM 1/2/00

61. An antisense molecule comprising the isolated nucleic acid of claim

62. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 25 to 27.

63. An antisense molecule comprising the isolated nucleic acid of claim 62.

64. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 28 to An antisense molecule comprising the isolated nucleic acid of claim 64.

66. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 31 to 33.

67. An antisense molecule comprising the isolated nucleic acid of claim 66.

68. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid accding to any one of claims 34 to 36.

69. An antisense molecule comprising the isolated nucleic acid of claim 68.

70. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 37 to 39.

71. An antisense molecule comprising the isolated nucleic acid of claim

72. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 40 to 41. \73. An antisense molecule comprising the isolated nucleic acid of claim 72. 9 4 9 *r 9 9 S* *i 9 S 9 99 9 9 9 9999 P:\OPER\MRO\14330-97.CLM 1/2/00 -26-

74. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 42 to 44. An antisense molecule comprising the isolated nucleic acid of claim 74.

76. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 45 to 47.

77. An antisense molecule comprising the isolated nucleic acid of claim 76.

78. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 48 to

79. An antisense molecule comprising the isolated nucleic acid of claim 78. 4,

80. An isolated nucleic acid that comprises a nucleotide sequence that is complementary to the nucleotide sequence of the nucleic acid according to any one of claims 51 to 53.

82. A method for preparing a recombinant vector including: providing the isolated nucleic acid according to any one of claims 13 to 81; and *o*o a vector; and (ii) reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.

83. The method according to claim 82 wherein the nucleic acid is prepared by a process comprising the method according to any one of claims 1 to The method according to claim 82 wherein the vector is a plasmid expression vector P:\OPER\MRO\14330-97.CLM 1/2/00 -27- selected from the group consisting of Bluescript SK+; a binary vector; and a vector containing a promoter that is operable in a plant cell. The method according to claim 84 wherein the promoter is a constitutive promoter.

86. A recombinant vector that comprises the isolated nucleic acid according to any one of claims 13 to 81.

87. The recombinant vector of claim 86 wherein said vector is capable of being replicated in a unicellular organism or a plant.

88. The recombinant vector according to claims 86 or 87 wherein the nucleic acid is in a form that is capable of being transcribed or translated in a unicellular organism or a plant.

89. The recombinant vector according to and one of claims 86 to 88 wherein said nucleic acid is prepared by a process comprising the method according to any one of claims 1 to

90. A method of decreasing the level of PPO activity in a plant tissue, which method includes: providing the isolated nucleic acid according to any one of claims 13 to 53 and a plant Y sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

91. A method of decreasing the level of PPO activity in a plant comprising expressing the .j isolated nucleic acid according to any one of claims 13 to 53 therein for a time and under conditions sufficient for cosuppression of endogenous PPO gene expression to occur.

92. The method according to claims 90 or 91 wherein said nucleic acid is prepared by a process comprising the method according to any one of claims 1 to The method according to any one of claims 90 to 92 wherein the nucleic acid is P:\OPER\MRO\14330-97.CLM 1/2/00 -28- introduced to the plant or a cell or tissue thereof by Agrobacterium-mediated transformation or by microprojectile bombardment.

94. A method of decreasing the level of PPO activity in a plant tissue comprising expressing the isolated nucleic acid according to any one of claims 54 to 81 therein for a time and under conditions sufficient for expression of an endogenous PPO gene to be reduced. The method of claim 94 wherein expressing the isolated nucleic acid according to any one of claims 54 to 81 comprises expressing antisense mRNA to target expression of an endogenous PPO gene in the plant tissue.

96. The method according to claims 94 or 95 wherein the nucleic acid is prepared by a process comprising the method according to any one of claims 1 to

97. The method according to any one of claims 94 to 96 further comprising introducing the isolated nucleic acid to plant tissue.

98. A method of increasing the level of PPO activity in plant tissue, which method includes providing the isolated nucleic acid according to any one of claims 13 to 53 and plant S tissue; and introducing said nucleic acid into said plant tissue to produce a transgenic plant. .99. A method of increasing the level of PPO activity in a plant tissue comprising expressing the isolated nucleic acid according to any one of claims 13 to 53 therein for a time and under conditions sufficient for expression of enzymatically-active protein to occur.

100. A method according to any one of claims 98 or 99 wherein the nucleic acid is prepared by a process comprising the method according to any one of claims 1 to

101. A transgenic plant which contains nucleic acid capable of expressing PPO or P:\OPER\MRO\14330-97.CLM -2/2/00 -29- modifying the expression of an endogenous PPO gene in said plant, wherein said nucleic acid comprises a nucleotide sequence that is substantially identical to the sequence of the nucleic acid according to any one of claims 13 to 81.

102. A method according to any one of claims 1 to 10 substantially as hereinbefore described with reference to the Figures and/or Examples. Dated this SECOND day of FEBRUARY, 2000 Commonwealth Scientific and Industrial Research Organisation by Davies Collison Cave Patent Attorneys for the Applicant