CA2290887A1

CA2290887A1 - Polyphenol oxidase genes from banana, tobacco and pineapple

Info

Publication number: CA2290887A1
Application number: CA002290887A
Authority: CA
Inventors: Simon Robinson
Original assignee: Individual
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1997-05-19
Filing date: 1998-05-19
Publication date: 1998-11-26
Also published as: CN1260836A; WO1998053080A1; AUPO684997A0; EP1012297A4; JP2001525677A; EP1012297A1; NZ501144A

Abstract

The present invention provides methods for preparing nucleic acids encoding polyphenol oxidase (PPO), fragments and derivatives tereof. The present invention also provides nucleic acids encoding banana, tobacco or pineapple PPO, or antisense to banana, tobacco or pineapple PPO, fragments and derivatives thereof. Vectors including such nucleic acids, methods of using such nucleic acids and transgenic plants are also provided.

Description

POLYPHENOL OXIDASE GENES FROM BANANA, TOBACCO ~ PINEAPPLE
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 PCTlAU92/00356 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. However, the method described in PCT/AU92/0035fi which was used to clone the PPO genes from apple and potato involved the use of an oligo dT reverse primer for polymerase chain reaction (PCR). Whilst the method is acceptable, in some tissues, it does not give rise to a strong band of the predicted size or else it gives rise to many additional products making it difficult to resolve the PPO fragment.
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.

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 source of a polypeptide having PPO activity, a first primer having a sequence corresponding to a first conserved region of PPO, and a second primer having a sequence corresponding to a second conserved region of PPO orientation;
isolating RNA from the source of polypeptide having PPO activity;
treating the RNA to construct copy DNA (cDNA) therefrom; and amplifying the cDNA so formed using the first and second primers.
Applicant has found that the method of the present invention, which involves the use of a second primer based on PPO, means that there is less likelihood that other (non-PPO) genes are amplified. Furthermore, the method of the present invention dramatically increases the amount of genuine product formed in most cases. Moreover, the added specificity provided by the second PPO-based primer makes it possible to clone PPO more readily from certain plants in which it was difficult to obtain a clone using one primer and oligo-dT.
In a preferred aspect of the present invention there is provided a method for preparing nucleic acid encoding banana, tobacco or pineapple PPO, fragments and derivatives thereof, which method includes providing a source of a polypeptide having banana, tobacco or pineapple PPO
activity, a first primer having a sequence corresponding to a first conserved region of banana, tobacco or pineapple PPO, and a second primer having a sequence corresponding to a second conserved region of banana, tobacco or pineapple PPO;
isolating RNA from the source of polypeptide having banana, tobacco or pineapple PPO activity;
treating the RNA to construct copy DNA (cDNA) therefrom; and amplifying the cDNA so formed using the first and second primers.
The terms "nucleic acid encoding banana/tobacco/pineapple PPO" and "banana/tobacco/pineapple PPO gene" as used herein should be understood to refer to a banana, tobacco and/or pineapple PPO gene or a sequence ' 5 substantially homologous therewith. For example, these terms include sequences which differ from the specific sequences given in the Examples hereto but which, because of the degeneracy of the genetic code, encode the same protein. Applicants have found that there are families of PPO genes in most plants. Thus, there are likely to be other PPO genes in banana, tobacco and/or pineapple, in addition to those which have been isolated. These could be cloned using the methods of the present invention. Thus, the terms "nucleic acid encoding banana/tobacco/pineapple PPO" and " bananaltobacco/pineapple PPO
gene" should be understood to include banana, tobacco and/or pineapple PPO
genes other than those specifc genes that have been isolated. The terms may also include presequences such as chioroplast transit sequence as well as sequences encoding mature PPO protein.
The term "derivative" as used herein includes nucleic acids that have been chemically or otherwise modified, for example mutated, or labelled, or nucleic acids incorporating a catalytic cleavage site.
The term "fragment" includes functionally active fragments of a PPO gene which are capable of altering expression of the PPO genes. Examples of alteration of the gene may include up-regulation or down-regulation of the gene, coding of the gene, transcription of the gene, binding of the gene or stability of the gene sequence.
The source of polypeptide having PPO activity is preferably a source of polypeptide having banana or tobacco or pineapple PPO activity. The source of polypeptide having banana PPO activity may be banana peel, preferably young banana peel. More preferably the peel of young banana fruit. The source of . polypeptide having tobacco PPO activity may be tobacco leaves, preferably young tobacco leaves. The source of polypeptide having pineapple PPO activity may be pineapple fruit, preferably the flesh of the pineapple fruit, more preferably the flesh of pineapple fruit exhibiting blackheart disorder.

The RNA may be isolated by any suitable method including extraction for example with a detergent such as CTAB, use of an oligo-dT spun column as described in PCT/AU92/00358 and PCT/AU96/00310 the entire disclosure of each application which is incorporated herein by reference, or use of a commercially available kit such as the PolyATtract 1000 system from Promega Corporation.
The step of treating the RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reverse transcriptase and an adapter primer to form cDNA.
The adapter primer may be an oligonucleotide adapter primer including the following sequence or part thereof:

5'-GACTCGAGTCGACATCGA -3' The step of treating the RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reverse transcriptase and a reverse primer to form cDNA.
The adapter primer may be replaced with a reverse primer having a sequence corresponding to a conserved region of PPO genes including the following sequence or part thereof:
REV2 :5'-GCCTGCAGTT[TC]TC[AG]TC[AGJTAGAA-3' The first primer has a sequence corresponding to a first conserved region of PPO. Preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to a first copper binding site of PPO. The second primer has a sequence corresponding to a second conserved region of PPO.
Preferably the second primer has a sequence corresponding to at least a portion of or in close proximity to a second copper binding site of PPO. More preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to one of the CuA or CuB binding sites of PPO, and the second primer has a sequence corresponding to at least a portion of or in close proximity to 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 of the following sequences or part thereof:
GEN8 : 5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3'.
GEN9 : 5'GCGAATTCTICA[TC]TG[TC)GCITA[TC]TG-3'.
GEN 10: 5'GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3'.
' S The second primer may include the following sequences or part thereof REV1 : 5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3'.
REV2 : 5'GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3'.
The cDNA may be amplified using the polymerase chain reaction (PCR).
Those skilled in the art will appreciate that if the Cu binding sites are internal, the nucleic acid isolated will be a fragment of the PPO gene lacking 3' and 5' termini. However, 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.
Accordingly, in a further aspect of the present invention there is provided a method for preparing nucleic acid encoding the 3' end of PPO, which method includes providing a source of polypeptide having PPO activity a primer in sense orientation; and an adapter primer;
isolating RNA from the source of polypeptide having PPO activity;
treating the RNA to construct cDNA therefrom; and amplifying the cDNA so formed using the primers.
In a further aspect of the present invention there is provided a method for preparing nucleic acid encoding the 5' end of PPO, which method includes providing a source of polypeptide having PPO activity, an anchor, primers in antisense orientation; and an anchor primer;
isolating RNA from the source of polypeptide having PPO activity;
treating the RNA to construct cDNA therefrom;

attaching the anchor to the 5' end of the cDNA so formed; and amplifying the cDNA using the primers.
The source of polypeptide having PPO activity is preferably a source of polypeptide having banana or tobacco or pineapple PPO activity. The source of polypeptide having banana PPO activity may be banana peel, preferably young banana peel. More preferably the peel of young banana fruit. The source of polypeptide having tobacco PPO activity may be tobacco leaves, preferably young tobacco leaves. The source of polypeptide having pineapple PPO activity may be pineapple fruit, preferably the flesh of the pineapple fruit, most preferably the flesh of pineapple fruit exhibiting blackheart disorder.
The RNA may be isolated by any suitable method including extraction for example with a detergent such as CTAB, use of an oligo-dT spun column as described in PCT/AU92/00356 or PCT/AU96/00310 the entire disclosure of each patent application which is incorporated herein by reference, or use of a commercially available kit such as the PolyATtract 1000 system from Promega Corporation.
The step of treating the RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reverse transcriptase and an adapter primer to form cDNA.
The adapter primer may be an oligonucleotide adapter primer including one of the following sequences or part thereof:
5'-GACTCGAGTCGACATCGATTTT -3' The adapter primer may be replaced with a reverse primer having a sequence corresponding to a conserved region of PPO genes including the following sequence or part thereof:
REV2 :5'-GCCTGCAGTT[TCJTC[AG]TC[AG]TAGAA-3' The primer in sense orientation may be a banana, tobacco or pineapple PPO specific primer.
The adapter primer may include the following sequence or part thereof:
5'-GACTCGAGTCGACATCGA-3'.
The primers in antisense orientation may be banana, tobacco or pineapple PPO specific primers.
The anchor may be of any suitable type. The anchor may be attached by . ligation for example using T4 RNA ligase. The anchor primer should be capable of hybridizing with the anchor.
The cDNA may be amplified using PCR.
Those skilled in the art will appreciate that using the methods of the present invention it is possible to determine the complete nucleic acid sequence of the PPO gene of interest 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 banana PPO or antisense to banana PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Fig. 1, 2, 3 or 4, fragments and derivatives thereof, and substantially homologous sequences.
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 Fig. 5, fi or 7 fragments and derivatives thereof, and substantially homologous sequences.
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. Preferably the nucleic acid has the sequence shown in Fig.

8, 9 or 10 fragments and derivatives thereof, and substantially homologous sequences.
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 banana PPO or antisense to banana PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.
In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding tobacco PPO
or antisense to tobacco PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.
In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding pineapple PPO
or antisense to pineapple PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof; and 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 cieavage 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 of the vector, 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, 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 banana PPO or antisense to banana PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.
In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.
In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding pineapple PPO or antisense to pineapple 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 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 banana, tobacco or pineapple PPO or antisense to banana, tobacco or pineapple PPO, fragments and derivatives thereof.
The microorganism may be a strain of Escherichia coli, for example E.coli DH5 has been found to be suitable.
5 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 banana PPO, a modified nucleic acid encoding banana PPO, or a nucleic acid antisense to banana PPO, 10 fragments and derivatives thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.
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 tobacco PPO, a modified nucleic acid encoding tobacco PPO, or a nucleic acid antisense to tobacco PPO, fragments and derivatives thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.
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 pineapple PPO, a modifed nucleic acid encoding pineapple PPO, or a nucleic acid antisense to pineapple 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 banana or tobacco or pineapple PPO or a functionally active fragment thereof. Alternatively the nucleic acid may encode banana or tobacco or pineapple PPO or a functionally active 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 by infection of the plant with an Aarobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles. Methods for transforming banana, tobacco or pineapple with Agrobacterium are known to those skilled in the art and are described in, for example, May et al., Biotechnology (1995) 13:486-492;
Michelmore et al., Plant Cell Reports (1987) 6:439-442, and Curtis et al., Journal of Experimental Botany (1994) 45:1141-1149, the entire disclosures of which one incorporated herein by reference. Methods for transforming banana, tobacco or pineapple by bombardment with DNA coated microprojectiles are known to those skilled in the art and are described in, for example, Sagi et al., Biotechnology (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 banana PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.
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 tobacco PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.
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 pineapple 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 info the plant may be by infection of the plant with an Agrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiies.
The plant may be of any suitable type. However the method is particularly applicable to banana, tobacco or pineapple.
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 banana PPO gene.
The plant may be of any suitable type. Preferably, the plant is banana.
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 tobacco PPO gene.
The plant may be of any suitable type. Preferably, the plant is tobacco.
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 pineapple PPO gene.
The plant may be of any suitable type. Preferably, the plant is pineapple.
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 banana PPO or antisense to banana PPO, fragments and derivatives thereof.
In a still further aspect of the present invention there is provided a plant CA 02290887 1999-m-is PCT/AU98/00362 , Received 19 May 1999 P:\OPER1MR0~.9ri-O()352.PCT - IBlS/99 vaccine including nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof.
In a still further aspect of the present invention there is provided a plant vaccine including nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof.
The present invention will now be more fully described with reference to the accompanying Examples. 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 BPP02 cDNA nucleotide sequence (SEQ ID NO: 1 ) and derived protein sequence (SEQ ID NO: 2) encoding part of a banana PPO protein.
FIGURE 2: The BPP08 cDNA nucleotide sequence (SEQ ID N0:3) and derived protein 1 S sequence (SEQ ID NO: 4) encoding part of a banana PPO protein.
FIGURE 3: The BANPP034 cDNA nucleotide sequence (SEQ ID NO:S) and derived protein sequence (SEQ ID NO: 6) encoding part of a banana PPO protein.
FIGURE 4: The BANPP035 cDNA nucleotide sequence (SEQ ID N0:7) and derived protein sequence (SEQ ID N0:8) encoding part of a banana PPO protein.
FIGURE 5: The TOBPP06 cDNA nucleotide sequence (SEQ ID NO: 9) and derived protein sequence (SEQ ID NO:10) encoding part of a tobacco PPO protein.
FIGURE 6 The TOBPP025 cDNA nucleotide sequence (SEQ ID NO:11) and derived protein sequence (SEQ ID N0:12) encoding part of a tobacco PPO protein.
FIGURE 7: The TOBPP026 cDNA nucleotide sequence (SEQ ID N0:13) and derived protein sequence (SEQ ID N0:14) encoding part of a tobacco PPO protein.
AMENDED S~~(Aiiticle 34) (IPEA/ALn CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 ' P'. \OPE R\MRO\98-00362. PCT - 18/5/99 FIGURE 8: The PINPP020 cDNA nucleotide sequence (SEQ ID NO:15) and derived protein sequence (SEQ ID N0:16) encoding part of a pineapple PPO protein.
FIGURE 9: The PINPP02 cDNA nucleotide sequence (SEQ ID N0:17) and derived protein sequence (SEQ ID N0:18) encoding part of a pineapple PPO protein.
FIGURE 10: The PINPPOFL cDNA nucleotide sequence (SEQ ID N0:19) and derived protein sequence (SEQ ID N0:20) encoding a pineapple PPO protein.
EXAMPLE 1: Cloning Banana Peel PPO genes Total RNA was isolated from the peel of young banana fruit. Fruit tissue (3g) was frozen and ground to a fine powder in liquid nitrogen with a coffee grinder then added to 20 ml of extraction buffer (2% hexadecyltrimethylammonium bromide (CTAB), 2%
polyvinyl pyrolidone, 100 mM Tris-CHI, pH 8.0, 25 mM EDTA, 2 M NaCI, 0.05% spermidine, 2%
~i-mercaptoethanol) at 65°C. The extract was mixed with 20 ml of chloroform / IAA then centrifuged for 20 minutes at 5,000 RPM and the aqueous phase was re-extracted with chloroform / IAA. The aqueous phase was filtered through Miracloth and 0.25 volumes of 10 M LiCI were added then the sample was incubated overnight at 4°C
before centrifuging for 20 minutes at 8,000 RPM. The supernatant was removed and the pellet was resuspended in 0.5 ml of 1 M NaCI, 0.5 % SDS, 10 mM Tris, pH 8.0, 1mM EDTA. The RNA was extracted once with an equal volume of chloroform / IAA and 2 volumes of ethanol was added. After incubation for 40 mins at -70°C the solution was centrifuged for 15 minutes at 10,000 RPM. The supernatant was removed and the pellet was rinsed with 80%
ethanol, drained and dried. The pellet was resuspended in 50 ~,L of sterile water.
First strand cDNA was synthesised from 10~,g total RNA with reverse transcriptase as described in Ref 2, utilising an oligosaccharide-dT primer adapter (Ref 1):
B26 (SEQ ID N0:32): (5'GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3').
AMENDED SHEET (Article 34) (IPEA/Ain CA 02290887 1999-m-is PCT/AU98/00362 ' ' , Received 19 May 1999 ' P:\OPER\MRO\9ri-(p362.POT - 18/5/99 Oligonucleotide primers were designed based on known plant PPO DNA sequences.
Comparison of a number of PPO sequences from a range of different plants allowed identification of the conserved regions of the gene, which are mostly in or near the regions which encode the two copper binding sites, CuA and CuB (2). Forward primers designed around the CuA site (GENB, GEN9 and GEN 10) and reverse primers designed around the CAB site (REV 1 and REV2) were synthesised:
GEN8 (SEQ ID N0:22): (5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3') GEN9 (SEQ ID N0:23): (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') GEN10 (SEQ ID NO:24):(5'-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3') REV1 (SEQ ID NO:25):(5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3') REV2 (SEQ ID NO:26):(5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3') The first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman (1) using GEN and REV primers, each at a final concentration of 1~,M (2). Amplification involved an initial program of 2 cycles of denaturation at 94 ° C for 1 min, annealing at 37 ° C for 2 min, a slow ramp to 72 ° C for 3 min, followed by 33 cycles of denaturation at 94 ° C for 1 min, annealing at 55 ° C for 1 min, and elongation at 72°C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised.
DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).
The purified DNA was cloned into Eco -RV-cut Bluescript SK+ vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E.
coli DHSa by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Two putative banana PPO clones (BPP02 and BPP08) were identified based on their homology to known plant PPO genes.
AMENDED SHEET (Article 34) (IPEA/Ai~

CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 P: \OPER\MRO\99-W 362. PCT - 19/5/99 The 3'-end of BPP02 was cloned using a primer designed to the sequence of BPP02:
BANBF (SEQ ID N0:27): (5'-GTTGCTCTTCTTAGGCTCGGCTTAC-3') at a final concentration of 1~M and a B25 adaptor primer:
B25:(SEQ ID N0:28):(5'GACTCGAGTCGACATCGA-3') at a final concentration of l~cM (ref 1). Amplification involved 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).
The purified DNA was cloned into Eco RV-cut Bluescript SK+ vector (Stratagene) which has been T-tailed with Taq Polymerase and the ligated DNA was introduced into E.coli DHSa by electroporation. Recombinant clones which had an insert of the predicted size (1150 bp) were selected and their DNA sequence was determined by automated sequencing.
Two putative banana PPO clones (BANPP034 and BANPP035) were identified based on their homology to know plant PPO genes. The sequence of BANPP034 was identical to that of BPP02.
EXAMPLE 2: Cloning Tobacco Leaf PPO genes Total RNA was isolated from young leaves (1-3cm long) of glasshouse grown plants.
Approximately 2 g of frozen leaf material was ground to a fine powder in liquid nitrogen then extracted in 15 ml of extraction buffer (50 mM Tris-HCI, pH 9.0, 150 mM LiCI, 5mM
EDTA, 5 % SDS and 0.6 % ~3-mercaptoethanol) by shaking vigorously in a 50 ml screw cap tube for 1-2 minutes.
Approximately 15 ml of phenol / chloroform / IAA (25: 24:1 ) was added and the homogenate AMENDED SHEET (Article 34) (IPEA/ALn mixed then centrifuged for 15 minutes at 5,000 RPM, 4°C. The upper aqueous phase was removed and re-extracted twice with phenol / chloroform / IAA and then once with chloroform / IAA and then centrifuged for 10 minutes at 5,000 RPM, 4°C. The supernatant was removed, LiCI was added to a final concentration of 2 M and the mixture was incubated overnight at 4°C.
After centrifuging for 10 minutes at 8,000 RPM, 4°C the supernatant was removed and the pellet was resuspended in 6 ml of 0.4 M LiCI then 2 ml of 8M LiCI was added and the mixture was incubated overnight at 4°C. The mixture was centrifuged for minutes at 8,000 RPM, 4°C, the supernatant was removed and the pellet was 10 resuspended in 0.5 ml of sterile water and centrifuged briefly to remove any insoluble material.
mRNA was isolated from the total RNA using a PolyATtract kit (Promega). First strand cDNA was synthesised from 10 pg total RNA or 2 ~,g mRNA with reverse transcriptase as described in Ref 2, utilising an oligo-dT primer adapter (Ref 1 ) B26 : (5'-GACTCGAGTCGACATCGATTTTIfTTTTTTTTTTTT-3') The first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman (1) using GEN and REV primers described in Example 1, each at a final concentration of 1 pM (2).
Amplification involved an initial program of 2 cycles of denaturation at 94° C for 1 min, annealing at 37° C for 2 min, a slow ramp to 72° C over 2 min and elongation at 72° C for 3 min, followed by 28 cycles of denaturation at 94° C
for 1 min, annealing at 55° C for 1 min, and elongation at 72° C for 3 min.
A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).
The purified DNA was cloned into Eco RV-cut Bluescript SK+ vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli DHSa by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Three putative tobacco PPO clones (TOBPP06, TOBPP025 and TOBPP026) were identified based on their homology to known plant PPO genes.
EXAMPLE 3: Cloning Pineapple PPO genes Mature pineapple fruit were treated to induce biackheart disorder by holding the fruit for 17 days at 12°C then for 4 days at 25°C. Flesh showing blackheart symptoms was dissected from the fruit, frozen in liquid nitrogen and ground to a fine powder in a pre-cooled coffee grinder. To isolate total RNA 10 g of the powder was ground in a mortar and pestle then extracted with 30 ml of homogenisation buffer (100mM Tris-HCI, pH9.0, 200mM NaCI, 15 mM EDTA, 0.5% sarkosyl and 1 % ~-mercaptoethanol), 30 ml of phenol and 6 ml of chloroform / IAA. The mixture was stirred in a beaker, 2.1 ml of 3M NaAc (pH
5.2) was added and the mixture was kept on ice for 15 minutes then centrifuged for 15 minutes at 8,000 RPM, 4°C. The upper aqueous phase was removed and an equal volume of isopropano! was added. The mixture was incubated for 30 minutes at -70°C then centrifuged for 20 minutes at 8,000 RPM, 4°C in Corex tubes. The supernatant was removed and the pellet was rinsed with 70%
ethanol and centrifuged for 5 minutes at 8,000 RPM, 4°C. The ethanol was removed and the pellet was air dried then resuspended in 0.75 ml sterile water and centrifuged to remove any insoluble material. LiCI was added to a final concentration of 3 M and the mixture was incubated overnight at -20°C
then centrifuged for 30 minutes at 15,000 RPM, 4°C. The pellet was rinsed with 70%
ethanol, centrifuged briefly, drained and air dried. The pellet was resuspended in 75 p.l sterile water and centrifuged to remove any insoluble material.
Oligonucleotide primers were designed based on known plant PPO DNA
sequences. Comparison of a number of PPO sequences from a range of different plants allowed identification of the conserved regions of the gene, which are mostly in or near the regions which encode the two copper binding sites, CuA
and CuB. Forward primers designed around the CuA site (GEN8, GEN9 and GEN 10) and reverse primers designed around the CuB site (REV1 and REV2) were synthesised GEN8 : (5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3') GEN9 : (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') GEN10 : (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') First strand cDNA was synthesised from 10 ~g total RNA with reverse transcriptase as described in Ref 2, utilising the REV2 primer REV2 : (5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3') The first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman (1 ) using the GEN and REV
primers described in Example 1, each at a final concentration of 1 ~M (2).
Amplification involved an initial program of 2 cycles of denaturation at 94° C for 1 min, annealing at 37° C for 2 min, a slow ramp to 72° C over 2 min and elongation at 72° C for 3 min, followed by 33 cycles of denaturation at 94°
C for 1 min, annealing at 55° C for 1 min, and elongation at 72° C for 3 min.
A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised.
DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).
The purified DNA was cloned into Eco RV-cut Bluescript SK+ vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli DHSa by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. A putative pineapple PPO clone (PINPP020) was identified based on its homology to known plant PPO genes.

First strand cDNA was also synthesised from 10 ~g total RNA with reverse transcriptase as described in Dry, I.B. and Robinson, S. P (1994), utilising an oligo-dT primer adapter (Ref 1 ) B26 : (5'-GACTCGAGTCGACATCGA TT-3') 5 This first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman, M.A. (1990) using GEN9 and GEN10 primers GEN9 : (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') GEN10 : (5'-GCGAATTCTTiCCIT[TA][TC]TGGAA(TC]TGGG-3') 10 at a final concentration of 1 p.M and a B25 adaptor primer B25 : (5'-GACTCGAGTCGACATCGA-3') at a final concentration of 0.1 ~.M (Frohman, M.A. (1990); Dry, I.B. and Robinson, S.P. (1994)) Amplification involved a program of 33 cycles of denaturation at 94°
C for 1 min, annealing at 55° C for 1 min, and elongation at 72° C for 3 min.
A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised.
DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).
The purified DNA was cloned into Eco RV-cut Bluescript SK+ vector (Stratagene}
which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli DHSa by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Two putative pineapple PPO clones (PINPP01 and PINPP02) were identified based on their homology to known plant PPO genes. The sequence of PINPP01 was found to be nearly identical to that of PINPP020.
The 5'-end of PINPP01 was obtained using a 5'-RACE system for rapid amplification of cDNA ends, Version 2.0, from GIBCO-BRL, according to the _ , CA 02290887 1999-ii-is pCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT ~ ISI5199 manufacturer's instructions. Specific oligonucleotide primers based on the sequences of PINPPO1 and PINPP02 were used:
PINE 1: (SEQ ID N0:29): 5-'ATATCACCTGTCGGTACATGACGGC-3' PINE 2: (SEQ ID N0:30):5'- GTGCCATTGTAGTCGAGGTCAATCA-3' S A number of clones were sequenced and one, SPINA, was found to be nearly identical to PINPPO1 in the overlapping regions.
A full-length pineapple cDNA clone was isolated using a primer designed to the 5'-end sequence of SPINA:
5PIN1: (SEQ ID NO:31):(5'-CCAGTGCCTGGTTTAGGTGTATTCAC-3') are used with the B25 adaptor primer as described above to amplify cDNA
prepared from blackheart-induced pineapple fruit RNA. Amplification involved a program of 33 cycles of denaturation at 94 ° C for 1 min, annealing at 55 ° C for 1 min, and elongation at 72 ° C for 3 mm.
A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gen and the bands of interest were excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).
The purified DNA was cloned into Eco RV-cut Bluescript SK+ vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E.coli DHSa by electroporation. Recombinant clones which had an insert of the predicted size (2.2kbp) were selected and their DNA sequence was determined by automated sequencing. A
pineapple PPO clone (PINPPOFL) was identified based on its homology to the PINPP020, PINPPO1 and 5 PINA clones. The sequence of PINPPOFL was found to be nearly identical to that of PINPP020, PINPPO1 and SPINA in the overlapping regions.
AMENDED SHEET (Article 34) (IPEA/ALn CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 P'\OPER\MRO\98-00362.PCT ~ 1815/99 GENERAL
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The 5 invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
Throughout this specification, unless the context requires otherwise the word "comprise", and 10 variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
REFERENCES
1. Frohman, MA (1990) in "PCR Protocols: A guide to Methods and Applications"
(MA
Innis, DH Gelfrand, JJ Sninsky and TJ White, eds) Academic Press, New York, pp 28-38.
2. Dry, IB and Robinson, SP (1994) "Molecular cloning and characterisation of grape berry polyphenol oxidase". Plant Mol. Biol. 26, 495-502.
AMENDED SHEET (Article 34) (IPEA/A~

CA 02290887 1999-m-is PCT/AU98/00362 .
Received 19 May 1999 A~.v21.i4SG6.SEQ- lRiS/99 SEQUENCE LISTING
<110> COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION
<120> Polyphenol oxidase genes from banana, tobacco and pineapple <130> PCT/AU98/00362 <140> PCT/AU98/00362 <141> 1998-05-19 <150> AU PP6849 <151> 1997-05-19 <160> 33 <170> PatentIn Ver. 2.0 <210> 1 <211> 582 <212> DNA
<213> banana <220>
<221> CDS
<222> (1)..(582) <400> 1 cac tgt gcg tat tgt gat ggc gcc tac gac cag atc ggc ttc ccc aac 48 His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gln Ile Gly Phe Pro Asn ctc gag ctc caa gtc cac aac tcc tgg ctc ttc ttc cct tgg cac cgc 96 Leu Glu Leu Gln Val His Asn Ser Trp Leu Phe Phe Pro Trp His Arg ttc tac ctc tac ttc cac gag agg atc ctc gga aag ctc ata ggc gac 144 Phe Tyr Leu Tyr Phe His Glu Arg Ile Leu Gly Lys Leu Ile Gly Asp gacact ttcgcc ctccctttc tggaac tgggacgcg cccggc ggcatg 192 AspThr PheAla LeuProPhe TrpAsn TrpAspAla ProGly GlyMet aagctg ccgtcg atctacgcc gaccct tcgtcctcg ctctat gacaag 240 LysLeu ProSer IleTyrAla AspPro SerSerSer LeuTyr AspLys tttcgc gacgcc aagcaccag ccgcca gtcctcgtc gacctc gactac 288 PheArg AspAla LysHisGln ProPro ValLeuVal AspLeu AspTyr aacgga accgac cctagtttc accgac gcagagcag atcgat cagaac 336 AsnGly ThrAsp ProSerPhe ThrAsp AlaGluGln IleAsp GlnAsn ctcaag atcatg taccggcag gtgatc tccaacggc aagacg ccgttg 384 LeuLys IleMet TyrArgGln ValIle SerAsnGly LysThr ProLeu AMENDED SHEET (Article 34) (IPEA/Ain CA 02290887 1999-11-18 ~ ' ~' .,~~T g/~H=3', .,=,s>
Received .I~'~ ~ ~ ~ '~yN.
.4:~: 144A6f.SEQ - 1815/99 ctcttctta ggctcgget taccgt gccggcgac aacccaaac cccggc 432 LeuPheLeu GlySerAla TyrArg AlaGlyAsp AsnProAsn ProGly gcgggctcg ctcgagaac atacca cacggcccc gtccacggg tggact 480 AlaGlySer LeuGluAsn IlePro HisGlyPro ValHisGly TrpThr ggcgacaga agccaaccc aatctc gaggacatg ggcaacttc tactcc 528 GlyAspArg SerGlnPro AsnLeu GluAspMet GlyAsnPhe TyrSer gcggggcgc gaccctatc ttcttc gcccaccat tcaaatgtc gatcgc 576 AlaGlyArg AspProIle PhePhe AlaHisHis SerAsnVal AspArg atgtgg MetTrp <210> 2 <211> 194 <212> PRT
<213> banana <400> 2 His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gln Ile Gly Phe Pro Asn Leu Glu Leu Gln Val His Asn Ser Trp Leu Phe Phe Pro Trp His Arg Phe Tyr Leu Tyr Phe His Glu Arg Ile Leu Gly Lys Leu Ile Gly Asp Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met Lys Leu Pro Ser Ile Tyr Ala Asp Pro Ser Ser Ser Leu Tyr Asp Lys Phe Arg Asp Ala Lys His Gln Pro Pro Val Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp Pro Ser Phe Thr Asp Ala Glu Gln Ile Asp Gln Asn Leu Lys Ile Met Tyr Arg Gln Val Ile Ser Asn Gly Lys Thr Pro Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro Gly Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp Thr Gly Asp Arg Ser Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Arg Asp Pro Ile Phe Phe Ala His His Ser Asn Val Asp Arg AMENDED SHEET (Article 34) (IPEA/AT.n _ , , CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 :1:'~14486fi.56Q- IY/Si99 Met Trp <210> 3 <211> 426 <212> DNA

<213> banana <220>

<221> CDS

<222> (1)..(426) <400> 3 ttg ccg tgg aattgggac gcgcccggc ggcatg aagctgccg tcg 48 ttt Leu Pro Trp AsnTrpAsp AlaProGly GlyMet LysLeuPro Ser Phe atc tac gac ccttcgtcc tcgctctat gacaag tttcgcgac gcc 96 gcc Ile Tyr Asp ProSerSer SerLeuTyr AspLys PheArgAsp Ala Ala aag cac ccg ccggtcctc gtcgacctc gactac aacggaacc gac 144 cag Lys His Pro ProValLeu ValAspLeu AspTyr AsnGlyThr Asp Gln cct agt acc gacgcagag cagatcgat cagaac ctcaagatc atg 192 ttc Pro Ser Thr AspAlaGlu GlnIleAsp GlnAsn LeuLysIle Met Phe tac cgg gtg atctccaac ggcaagacg ccgttg ctcttctta ggc 240 cag Tyr Arg Val IleSerAsn GlyLysThr ProLeu LeuPheLeu Gly Gln tcg get cgt gccggcgac aacccaaac cccggc gcgggctcg ctc 288 tac Ser Ala Arg AlaGlyAsp AsnProAsn ProGly AlaGlySer Leu Tyr gag aac cca cacggcccc gtccacggg tggact ggcgacaga agc 336 ata Glu Asn Pro HisGlyPro ValHisGly TrpThr GlyAspArg Ser Ile caa ccc ctc gaggacatg ggcaacttc tactcc gcggggcgc gac 384 aat Gln Pro Leu GluAspMet GlyAsnPhe TyrSer AlaGlyArg Asp Asn cct atc ttc gcccaccat tcaaatgtc gatagc atgtgg 426 ttc Pro Ile Phe AlaHisHis SerAsnVal AspSer MetTrp Phe <210> 4 <211> 142 <212> PRT

<213> banana <400> 4 Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met Lys Leu Pro Ser AMENDED SHEET (Article 34) (IPEA/ALn _ CA 02290887 1999-11-18 ' :~:.,i.,asFS.seQ-ixnw~ ReCelved 19 May 1999 Ile Tyr Ala Asp Pro Ser Ser Ser Leu Tyr Asp Lys Phe Arg Asp Ala Lys His Gln Pro Pro Val Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp Pro Ser Phe Thr Asp Ala Glu Gln Ile Asp Gln Asn Leu Lys Ile Met Tyr Arg Gln Val Ile Ser Asn Gly Lys Thr Pro Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro Gly Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp Thr Gly Asp Arg Ser Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Arg Asp Pro Ile Phe Phe Ala His His Ser Asn Val Asp Ser Met Trp <210> 5 <211> 925 <212> DNA
<213> banana <220>
<221> CDS
<222> (2) . . (853) <400> 5 g ttg ctc ttc tta ggc tcg get tac cgt gcc ggc gac aac cca aac cee 49 Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro ggc gcg ggc tcg ctc gag aac ata cca cac ggc ccc gtc cac ggg tgg 97 Gly Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp act ggc gac aga aac caa ccc aat ctc gag gac atg ggc aac ttc tac 145 Thr Gly Asp Arg Asn Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr tcc gcg ggg cgc gac cct atc ttc ttc gcc cac cat tca aac gtc gac 193 Ser Ala Gly Arg Asp Pro Ile Phe Phe Ala His His Ser Asn Val Asp cgc atg tgg tac ttg tgg aag aag ctc ggc ggg aag cat cag gac ttt 241 Arg Met Trp Tyr Leu Trp Lys Lys Leu Gly Gly Lys His Gln Asp Phe aac gat aag gac tgg ctc aac acc acc ttc ctc ttc tac gac gag aat 289 Asn Asp Lys Asp Trp Leu Asn Thr Thr Phe Leu Phe Tyr Asp Glu Asn get gac tta gtt cga gtc acc ctc aag gac tgc ttg cag ccg gag tgg 337 Ala Asp Leu Val Arg Val Thr Leu Lys Asp Cys Leu Gln Pro Glu Trp AMENDED SHEET (Article 34) (IPEA/Ain CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 .4: L 1.14riC~SEQ - I 8!5!99 ctt cgt tac gat tac caa gac gtc gag atc ccg tgg ctg aag acc cgg 385 Leu Arg Tyr Asp Tyr Gln Asp Val Glu Ile Pro Trp Leu Lys Thr Arg ccg act ccc aaa gcc ttg aag gcg cag aaa acc gca gcg aaa aca ctg 433 Pro Thr Pro Lys Ala Leu Lys Ala Gln Lys Thr Ala Ala Lys Thr Leu aaa get aca gca gag acg ccg ttc ccg gtg acg ctg caa tcc gcg gtg 481 Lys Ala Thr Ala Glu Thr Pro Phe Pro Val Thr Leu Gln Ser Ala Val agc acg acg gtg agg agg ccc aag gta tcg agg agc ggc aag gag aag 529 Ser Thr Thr Val Arg Arg Pro Lys Val Ser Arg Ser Gly Lys Glu Lys gaa gag gaa gag gag gtc ctc atc gtg gag ggg atc gag ttc gac cgc 577 Glu Glu Glu Glu Glu Val Leu Ile Val Glu Gly Ile Glu Phe Asp Arg gac tac ttc ata aag ttc gac gtc ttc gtg aac gcc acc.gag ggt gag 625 Asp Tyr Phe Ile Lys Phe Asp Val Phe Val Asn Ala Thr Glu Gly Glu ggc atc acg ccg ggc gcc agc gag ttc gcg ggc agc ttc gtc aac gtc 673 Gly Ile Thr Pro Gly Ala Ser Glu Phe Ala Gly Ser Phe Val Asn Val ccg cac aag cac aag cac agc aag aag gag aag aag ctg aag acg agg 721 Pro His Lys His Lys His Ser Lys Lys Glu Lys Lys Leu Lys Thr Arg ctc tgc ctg ggg atc act gac ctg ctc gag gac atc ggg gcg gag gac 769 Leu Cys Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp gac gac agc gtg ctc gtc acc atc gtc ccg aaa gcc gga aag ggc aag 817 Asp Asp Ser Val Leu Val Thr Ile Val Pro Lys Ala Gly Lys Gly Lys gtg tcg gtc gcc ggc ctc cgc atc gat ttc cca aat tgaagtaata 863 Val Ser Val Ala Gly Leu Arg Ile Asp Phe Pro Asn ctatatattt ctactaccta tcaaggaaaa taaaagccgc accatcgtaa caaaaaaaaa 923 as 925 <210> 6 <211> 284 <212> PRT
<213> banana <400> 6 Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro Gly Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp AMENDED SHEET (Article 34) (IPEA/Ain CA 02290887 1999-ii-is pCT/AU98/00362 Received 19 May 1999 .a ~maasc~s.sEQ- ~aism Thr Gly Asp Arg Asn Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Arg Asp Pro Ile Phe Phe Ala His His Ser Asn Val Asp Arg Met Trp Tyr Leu Trp Lys Lys Leu Gly Gly Lys His Gln Asp Phe Asn Asp Lys Asp Trp Leu Asn Thr Thr Phe Leu Phe Tyr Asp Glu Asn Ala Asp Leu Val Arg Val Thr Leu Lys Asp Cys Leu Gln Pro Glu Trp Leu Arg Tyr Asp Tyr Gln Asp Val Glu Ile Pro Trp Leu Lys Thr Arg Pro Thr Pro Lys Ala Leu Lys Ala Gln Lys Thr Ala Ala Lys Thr Leu Lys Ala Thr Ala Glu Thr Pro Phe Pro Val Thr Leu Gln Ser Ala Val Ser Thr Thr Val Arg Arg Pro Lys Val Ser Arg Ser Gly Lys Glu Lys Glu Glu Glu Glu Glu Val Leu Ile Val Glu Gly Ile Glu Phe Asp Arg Asp Tyr Phe Ile Lys Phe Asp Val Phe Val Asn Ala Thr Glu Gly Glu Gly Ile Thr Pro Gly Ala Ser Glu Phe Ala Gly Ser Phe Val Asn Val Pro His Lys His Lys His Ser Lys Lys Glu Lys Lys Leu Lys Thr Arg Leu Cys Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp Asp Asp Ser Val Leu Val Thr Ile Val Pro Lys Ala Gly Lys Gly Lys Val Ser Val Ala Gly Leu Arg Ile Asp Phe Pro Asn <210> 7 <211> 960 <212> DNA
<213> banana <220>
<221> CDS
<222> (2)..(853) <400> 7 AMENDED SHEET (Article 34) (IPEA/Ain , ' ' PCT/AU98/00362 Received 19 May 1999 .4.'~ Li4866.SEQ - I ~/Si99 g ttg ctc ttc tta ggc tcg get tac cgt gcc ggt gac cag cct aac ccc 49 Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Gln Pro Asn Pro ggc gcg gga tcc atc gag aac atg ccg cac aac aac gtg cac ttg tgg 97 Gly Ala Gly Ser Ile Glu Asn Met Pro His Asn Asn Val His Leu Trp acc ggc gac cgc acc cag ccc aac ttc gag aac atg ggc acc ttc tac 145 Thr Gly Asp Arg Thr Gln Pro Asn Phe Glu Asn Met Gly Thr Phe Tyr gcg gcg gcg cgc gac ccc atc ttc ttc gcc cac cac gcc aac atc gac 193 Ala Ala Ala Arg Asp Pro Ile Phe Phe Ala His His Ala Asn Ile Asp cga atg tgg tac ctg tgg aag aag ctc agc agg aag cac cag gac ttc 241 Arg Met Trp Tyr Leu Trp Lys Lys Leu Ser Arg Lys His Gln Asp Phe aat gac tcg gac tgg ctc aaa get tcc ttc ctc ttc tac gac gag aac 289 Asn Asp Ser Asp Trp Leu Lys Ala Ser Phe Leu Phe Tyr Asp Glu Asn gcc gac tta gtt cgg gtc acg gtc aag gac tgc ttg gag acc gag tgg 337 Ala Asp Leu Val Arg Val Thr Val Lys Asp Cys Leu Glu Thr Glu Trp ctg cgc tac acg tac caa gac gtg aag atc cca tgg gcg aac acc cga 385 Leu Arg Tyr Thr Tyr Gln Asp Val Lys Ile Pro Trp Ala Asn Thr Arg ccg act ccc aag ctc gcc aag gcg agg aaa gcc ggc agc aga tcg ctg 433 Pro Thr Pro Lys Leu Ala Lys Ala Arg Lys Ala Gly Ser Arg Ser Leu aaa gcc acc gcg gag gtg cag ttc cct gtg acg ctg gaa tcc ccg gtc 481 Lys Ala Thr Ala Glu Val Gln Phe Pro Val Thr Leu Glu Ser Pro Val aaa gtg acg gtg aag agg ccc aag gtg ggg agg agc ggc aag gag aag 529 Lys Val Thr Val Lys Arg Pro Lys Val Gly Arg Ser Gly Lys Glu Lys gaa gat gag gag gag ata ctc ata gtg gag ggg atc gag ttc gac cgc 577 Glu Asp Glu Glu Glu Ile Leu Ile Val Glu Gly Ile Glu Phe Asp Arg gac tac ttc atc aag ttc gac gtc ttc gtg aac gcg acg gag ggc gac 625 Asp Tyr Phe Ile Lys Phe Asp Val Phe Val Asn Ala Thr Glu Gly Asp ggc atc acg gcc ggg gcc agt gag ttc gcc ggc agc ttc gtg aac gtc 673 Gly Ile Thr Ala Gly Ala Ser Glu Phe Ala Gly Ser Phe Val Asn Val ccg cac aag cac aag cac cgc aag gat gag aat aag ctg aag acg agg 721 Pro His Lys His Lys His Arg Lys Asp Glu Asn Lys Leu Lys Thr Arg ctg tgt ctg gga atc acc gac ctg ctc gag gac atc ggc gcg gag gac 769 AMENDED SHEET (Article 34) (IPEA/Ain CA 02290887 1999-m-is PCT/AU98/00362 " Received 19 May 1999 P.-..~~Li.i3Ef.SEQ - IfS/5/99 Leu Cys Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp gac gac agc gtg ctc gtc acc atc gtg ccg aag gca ggc aaa gga aag 817 Asp Asp Ser Val Leu Val Thr Ile Val Pro Lys Ala Gly Lys Gly Lys gtg tcc gtc ggc ggt ctt cgg att gac ttt tcc aag tgaggaaata 863 Val Ser Val Gly Gly Leu Arg Ile Asp Phe Ser Lys aaagaattca cgtgccgtgc ctgctttcaa tgtacgaata aaataagagt gcatcatcac 923 cgaccatggt tctactttaa aaaaaaaaaa aaaaaaa 960 <210> 8 <211> 284 <212> PRT
<213> banana <400> 8 Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Gln Pro Asn Pro Gly Ala Gly Ser Ile Glu Asn Met Pro His Asn Asn Val His Leu Trp Thr Gly Asp Arg Thr Gln Pro Asn Phe Glu Asn Met Gly Thr Phe Tyr Ala Ala Ala Arg Asp Pro Ile Phe Phe Ala His His Ala Asn Ile Asp Arg Met Trp Tyr Leu Trp Lys Lys Leu Ser Arg Lys His Gln Asp Phe Asn Asp Ser Asp Trp Leu Lys Ala Ser Phe Leu Phe Tyr Asp Glu Asn Ala Asp Leu Val Arg Val Thr Val Lys Asp Cys Leu Glu Thr Glu Trp Leu Arg Tyr Thr Tyr Gln Asp Val Lys Ile Pro Trp Ala Asn Thr Arg Pro Thr Pro Lys Leu Ala Lys Ala Arg Lys Ala Gly Ser Arg Ser Leu Lys Ala Thr Ala Glu Val Gln Phe Pro Val Thr Leu Glu Ser Pro Val Lys Val Thr Val Lys Arg Pro Lys Val Gly Arg Ser Gly Lys Glu Lys Glu Asp Glu Glu Glu Ile Leu Ile Val Glu Gly Ile Glu Phe Asp Arg Asp Tyr Phe Ile Lys Phe Asp Val Phe Val Asn Ala Thr Glu Gly Asp AMENDED SHEET (Article 34) (IPEA/Ain ' ' PCT/AU98/00362 Received 19 May 1999 .4:4'.14.1966. SEQ - 15/5199 _9_ Gly Ile Thr Ala Gly Ala Ser Glu Phe Ala Gly Ser Phe Val Asn Val Pro His Lys His Lys His Arg Lys Asp Glu Asn Lys Leu Lys Thr Arg Leu Cys Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp Asp Asp Ser Val Leu Val Thr Ile Val Pro Lys Ala Gly Lys Gly Lys Val Ser Val Gly Gly Leu Arg Ile Asp Phe Ser Lys <210> 9 <211> 545 <212> DNA
<213> tobacco <220>
<221> CDS
<222> (1) . . (543) <400> 9 gat ccg acg ttt gcg ttg cca tat tgg aac tgg gat cat cca aag ggc 48 Asp Pro Thr Phe Ala Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly atg cgt ttg cca cac atg ttt gat caa cca aac gtg tac cct gat ctt 96 Met Arg Leu Pro His Met Phe Asp Gln Pro Asn Val Tyr Pro Asp Leu tac gat cca aga cgt aac caa gaa cac cgc ggt tct gta atc atg gac 144 Tyr Asp Pro Arg Arg Asn Gln Glu His Arg Gly Ser Val Ile Met Asp ctt ggt cat ttt ggt caa gac gtg aaa gga act gac ttg caa atg atg 192 Leu Gly His Phe Gly Gln Asp Val Lys Gly Thr Asp Leu Gln Met Met agc aat aac ctt act cta atg tat cgt caa atg att acc aat tca cca 240 Ser Asn Asn Leu Thr Leu Met Tyr Arg Gln Met Ile Thr Asn Ser Pro tgt cca caa ctc ttt ttc ggt aag cca tat tgt acg gaa gtt gga ccc 288 Cys Pro Gln Leu Phe Phe Gly Lys Pro Tyr Cys Thr Glu Val Gly Pro aaa cca ggg cag gga get att gaa aac atc cct cat act cct gtc cac 336 Lys Pro Gly Gln Gly Ala Ile Glu Asn Ile Pro His Thr Pro Val His att tgg gtt ggt agt aag cct aat gag aat aac tgt aaa aac ggt gaa 384 Ile Trp Val Gly Ser Lys Pro Asn Glu Asn Asn Cys Lys Asn Gly Glu gat atg gga aat ttc tat tca get ggt aag gat cct get ttc tat agt 432 Asp Met Gly Asn Phe Tyr Ser Ala Gly Lys Asp Pro Ala Phe Tyr Ser AMENDED SHEET (Article 34) (IPEA/Atn , , CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 A.Ll.l:lyfi(,.SEQ - Iri/5/99 cac cat gca aat gta gat cgc atg tgg aca ata tgg aaa aca tta gga 480 His His Ala Asn Val Asp Arg Met Trp Thr Ile Trp Lys Thr Leu Gly gga aaa cgc aag gac atc aac aag cca gat tat ttg aac act gag ttc 528 Gly Lys Arg Lys Asp Ile Asn Lys Pro Asp Tyr Leu Asn Thr Glu Phe ttt ttc tac gac gaa as 545 Phe Phe Tyr Asp Glu <210> 10 <211> 181 <212> PRT
<213> tobacco <400> 10 Asp Pro Thr Phe Ala Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly Met Arg Leu Pro His Met Phe Asp Gln Pro Asn Val Tyr Pro Asp Leu Tyr Asp Pro Arg Arg Asn Gln Glu His Arg Gly Ser Val Ile Met Asp Leu Gly His Phe Gly Gln Asp Val Lys Gly Thr Asp Leu Gln Met Met Ser Asn Asn Leu Thr Leu Met Tyr Arg Gln Met Ile Thr Asn Ser Pro Cys Pro Gln Leu Phe Phe Gly Lys Pro Tyr Cys Thr Glu Val Gly Pro Lys Pro Gly Gln Gly Ala Ile Glu Asn Ile Pro His Thr Pro Val His Ile Trp Val Gly Ser Lys Pro Asn Glu Asn Asn Cys Lys Asn Gly Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Lys Asp Pro Ala Phe Tyr Ser His His Ala Asn Val Asp Arg Met Trp Thr Ile Trp Lys Thr Leu Gly Gly Lys Arg Lys Asp Ile Asn Lys Pro Asp Tyr Leu Asn Thr Glu Phe Phe Phe Tyr Asp Glu <210> 11 <211> 673 <212> DNA
<213> tobacco AMENDED SHEET (Article 34) (IPEA/AU) , ' ' PCT/AU98/00362 . Received 19 May 1999 A.\21.~.186C.SEQ- IS/5/99 <220>
<221> CDS
<222> (3)..(671) <400> 11 tg cac tgt gcg tat tgc aac ggt get tac aaa att ggt ggc aaa gag 47 His Cys Ala Tyr Cys Asn Gly Ala Tyr Lys Ile Gly Gly Lys Glu tta caa gtc cat ttc tcg tgg ctt ttt ttc cct ttt cat aga tgg tac 95 Leu Gln Val His Phe Ser Trp Leu Phe Phe Pro Phe His Arg Trp Tyr ttg tac ttc tat gaa aga atc ttg ggc tct tta att aat gat cct act 143 Leu Tyr Phe Tyr Glu Arg Ile Leu Gly Ser Leu Ile Asn Asp Pro Thr ttt ggt ttg cca tat tgg aac tgg gac cat cca aag ggc atg cgt ata 191 Phe Gly Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly Met Arg Ile cct ccc atg ttc gat cgt gaa ggg tct tcc ctt tac gac gaa aaa cgt 239 Pro Pro Met Phe Asp Arg Glu Gly Ser Ser Leu Tyr Asp Glu Lys Arg aac caa agt cac cgt aat gga acc ata att gat ctt ggt cat ttc ggt 287 Asn Gln Ser His Arg Asn Gly Thr Ile Ile Asp Leu Gly His Phe Gly caa gaa gtc caa aca act caa ctg cag cag atg act aat aac tta act 335 Gln Glu Val Gln Thr Thr Gln Leu Gln Gln Met Thr Asn Asn Leu Thr ata atg tat cgt caa atg ata act aat get cct tgc ccc ttg ctc ttc 383 Ile Met Tyr Arg Gln Met Ile Thr Asn Ala Pro Cys Pro Leu Leu Phe ttt ggt cag cct tac cct cta gga act gat ccc agt cca ggg atg ggc 431 Phe Gly Gln Pro Tyr Pro Leu Gly Thr Asp Pro Ser Pro Gly Met Gly act att gaa aac atc cct cat act cct gtc cac att tgg gtt ggt agt 479 Thr Ile Glu Asn Ile Pro His Thr Pro Val His Ile Trp Val Gly Ser agg ctt gat gag aat aat acg aaa cac ggt gag gat atg ggt aat ttt 527 Arg Leu Asp Glu Asn Asn Thr Lys His Gly Glu Asp Met Gly Asn Phe tac tcg gcc ggt tta gac ccg ctt ttc tat tcc cat cac gcc aat gtg 575 Tyr Ser Ala Gly Leu Asp Pro Leu Phe Tyr Ser His His Ala Asn Val gac cgg atg tgg tcc gag tgg aaa gcc tta gga ggg aaa aga agg gat 623 Asp Arg Met Trp Ser Glu Trp Lys Ala Leu Gly Gly Lys Arg Arg Asp ctc acg cac aaa gat tgg ttg aac tcc gag ttc ttt ttc tac gat gaa 671 Leu Thr His Lys Asp Trp Leu Asn Ser Glu Phe Phe Phe Tyr Asp Glu AMENDED SHEET (Article 34) (IPEA/A~

. ' ' PCT/AU98/00362 ~.~~~~~ ~LQ g 5 ~ Received 19 May 1999 as 673 <210> 12 <211> 223 <212> PRT
<213> tobacco <400> 12 His Cys Ala Tyr Cys Asn Gly Ala Tyr Lys Ile Gly Gly Lys Glu Leu Gln Val His Phe Ser Trp Leu Phe Phe Pro Phe His Arg Trp Tyr Leu Tyr Phe Tyr Glu Arg Ile Leu Gly Ser Leu Ile Asn Asp Pro Thr Phe Gly Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly Met Arg Ile Pro Pro Met Phe Asp Arg Glu Gly Ser Ser Leu Tyr Asp Glu Lys Arg Asn Gln Ser His Arg Asn Gly Thr Ile Ile Asp Leu Gly His Phe Gly Gln Glu Val Gln Thr Thr Gln Leu Gln Gln Met Thr Asn Asn Leu Thr Ile Met Tyr Arg Gln Met Ile Thr Asn Ala Pro Cys Pro Leu Leu Phe Phe Gly Gln Pro Tyr Pro Leu Gly Thr Asp Pro Ser Pro Gly Met Gly Thr Ile Glu Asn Ile Pro His Thr Pro Val His Ile Trp Val Gly Ser Arg Leu Asp Glu Asn Asn Thr Lys His Gly Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Leu Asp Pro Leu Phe Tyr Ser His His Ala Asn Val Asp Arg Met Trp Ser Glu Trp Lys Ala Leu Gly Gly Lys Arg Arg Asp Leu Thr His Lys Asp Trp Leu Asn Ser Glu Phe Phe Phe Tyr Asp Glu <210> 13 <211> 685 <212> DNA
<213> tobacco <220>
<221> CDS
<222> (3)..(683) AMENDED SHEET (Article 34) (IPEA/AL~

CA 02290887 1999-ii-is PCT/AU98/00362 Received 19 May 1999 A: L 14J866. SEQ - 18/5199 <400>

tg cattgt gcg tgcaac gettac 47 tat gat aca atg ggt gac caa aag HisCys AlaTyr CysAsn AlaTyr Asp Thr Met Gly Asp Gln Lys ttacaagtt caccaa tcgtgg cttttcttc ccgttt catagatgg tac 95 LeuGlnVal HisGln SerTrp LeuPhePhe ProPhe HisArgTrp Tyr ttgtacttc tacgag agaatc ttgggctcc ctcatc gatgatcca act 143 LeuTyrPhe TyrGlu ArgIle LeuGlySer LeuIle AspAspPro Thr tttgetctg ccatat tggaac tgggaccat ccaagc ggcatgcgt ttg 191 PheAlaLeu ProTyr TrpAsn TrpAspHis ProSer GlyMetArg Leu cctgetatg ttcgat gtcgaa ggttcttcc ctctac gatgcaaga cgt 239 ProAlaMet PheAsp ValGlu GlySerSer LeuTyr AspAlaArg Arg aatccacat gtccgt aatgga accataatc gatctt ggttttttc ggt 287 AsnProHis ValArg AsnGly ThrIleIle AspLeu GlyPhePhe Gly gatgaagtc aaaact aatgaa atacagatg ataact aacaactta att 335 AspGluVal LysThr AsnGlu IleGlnMet IleThr AsnAsnLeu Ile ctaatgtat cgtcaa atgata actaatget ccatgc ccgctgttg ttc 383 LeuMetTyr ArgGln MetIle ThrAsnAla ProCys ProLeuLeu Phe ttc gga gag cct tac aga ttc gga tct aaa ccc aat ccg ggg cag gga 431 Phe Gly Glu Pro Tyr Arg Phe Gly Ser Lys Pro Asn Pro Gly Gln Gly accatt gaaaac attcctcat actccg gttcacatt tggact ggtact 479 ThrIle GluAsn IleProHis ThrPro ValHisIle TrpThr GlyThr gtgcgg tgtacg gatttgggt aattgt gtgccatca tacggt gaggat 527 ValArg CysThr AspLeuGly AsnCys ValProSer TyrGly GluAsp atgggt aatttc tactcaget ggttta gacccagtt ttttac agccac 575 MetGly AsnPhe TyrSerAla GlyLeu AspProVal PheTyr SerHis cacgcc aatgtg gaccgcatg tggaat gaatggaaa gcacta ggaggg 623 HisAla AsnVal AspArgMet TrpAsn GluTrpLys AlaLeu GlyGly aaaaga agggat ctcacagac aatgat tggttaaac tcggag ttcttt 671 LysArg ArgAsp LeuThrAsp AsnAsp TzpLeuAsn SerGlu PhePhe ttctac gacgaa as 685 PheTyr AspGlu AMENDED SHEET (Article 34) (IPEA/ALn CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 A:~2t4SS66.SEQ - I8/5/99 <210> 14 <211> 227 <212> PRT
<213> tobacco <400> 14 His Cys Ala Tyr Cys Asn Asp Ala Tyr Thr Met Gly Asp Gln Lys Leu Gln Val His Gln Ser Trp Leu Phe Phe Pro Phe His Arg Trp Tyr Leu Tyr Phe Tyr Glu Arg Ile Leu Gly Ser Leu Ile Asp Asp Pro Thr Phe Ala Leu Pro Tyr Trp Asn Trp Asp His Pro Ser Gly Met Arg Leu Pro Ala Met Phe Asp Val Glu Gly Ser Ser Leu Tyr Asp Ala Arg Arg Asn Pro His Val Arg Asn Gly Thr Ile Ile Asp Leu Gly Phe Phe Gly Asp Glu Val Lys Thr Asn Glu Ile Gln Met Ile Thr Asn Asn Leu Ile Leu Met Tyr Arg Gln Met Ile Thr Asn Ala Pro Cys Pro Leu Leu Phe Phe Gly Glu Pro Tyr Arg Phe Gly Ser Lys Pro Asn Pro Gly Gln Gly Thr Ile Glu Asn Ile Pro His Thr Pro Val His Ile Trp Thr Gly Thr Val Arg Cys Thr Asp Leu Gly Asn Cys Val Pro Ser Tyr Gly Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Leu Asp Pro Val Phe Tyr Ser His His Ala Asn Val Asp Arg Met Trp Asn Glu Trp Lys Ala Leu Gly Gly Lys Arg Arg Asp Leu Thr Asp Asn Asp Trp Leu Asn Ser Glu Phe Phe Phe Tyr Asp Glu <210> 15 <211> 670 <212> DNA
<213> pineapple <220>
<221> CDS
AMENDED SHEET (Article 34) (IPEA/AIn . PCT/AU98/00362 ' Received 19 May 1999 .A.~~L1.S~6(.SEQ- IS%5/99 <222> (3) .
. (668) <400> 15 tg cat tgt tgc 47 gcg tac gac ggc gcg tat gac caa atc ggc ttc ccc His Cys Ala Cys Gly Tyr Asp Ala Tyr Asp Gln Ile Gly Phe Pro gat ctc gag cagatc cacaactcg tggctcttc tttccttgg cac 95 atc Asp Leu Glu GlnIle HisAsnSer TrpLeuPhe PheProTxp Hi:, Ile cgg ttc tac tacttc aacgagcgc atactcggg aaacttatc ggc 143 ctc Arg Phe Tyr TyrPhe AsnGluArg IleLeuGly LysLeuIle Gly Leu gac gac acg gcgctg cctttctgg aactgggac gcgccgggg ggc 191 ttc Asp Asp Thr AlaLeu ProPheTrp AsnTrpAsp AlaProGly Gly Phe atg cag ttc ccg tct atc tac acg gac cct tca tcc tcg cta tat gac 239 Met Gln Phe Pro Ser Ile Tyr Thr Asp Pro Ser Ser Ser Leu Tyr Asp aag ctg cgt gat gcg aag cac cag ccg ccg act ttg att gac ctc gac 287 Lys Leu Arg Asp Ala Lys His Gln Pro Pro Thr Leu Ile Asp Leu Asp 80 g5 90 95 tac aat ggc acc gat cct acc ttc tcc cct gaa gaa cag att aac cac 335 Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln Ile Asn His aac ctc gcc gtc atg tac cga cag gtg ata tcc agt gga aag aca cca 383 Asn Leu Ala Val Met Tyr Arg Gln Val Ile Ser Ser Gly Lys Thr Pro gag ctg ttt atg ggc tca gcg tac cgc gcc ggt gac cag cct gac ccc 431 Glu Leu Phe Met Gly Ser Ala Tyr Arg Ala Gly Asp Gln Pro Asp Pro ggc gca ggt tct gta gag cag aag ccg cac ggc ccg gtg cat gtg tgg 479 Gly Ala Gly Ser Val Glu Gln Lys Pro His Gly Pro Val His Val Trp aca ggt gat cgc aac cag ccc aat cgc gaa gac atg ggc acg ctc tac 527 Thr Gly Asp Arg Asn Gln Pro Asn Arg Glu Asp Met Gly Thr Leu Tyr tcg gcg gcg tgg gac ccc gtt ttt ttc gca cac cac ggc aac atc gac 575 Ser Ala Ala Trp Asp Pro Val Phe Phe Ala His His Gly Asn Ile Asp cgc atg tgg tac gtg tgg agg aac ctt ggc ggc aag cac cgc aac ttc 623 Arg Met Trp Tyr Val Trp Arg Asn Leu Gly Gly Lys His Arg Asn Phe acc gac ccc gac tgg ctc aac gcg tcc ttc ctg ttc tac gac gaa as 670 Thr Asp Pro Asp Trp Leu Asn Ala Ser Phe Leu Phe Tyr Asp Glu <210> 16 AMENDED SHEET (Article 34) (IPEA/ALn ., , CA 02290887 1999-m-is PCT/AU98/00362 .4 maasc~.sEC~ - iaisi9v ReCelved 19 May 1999 <211> 222 <212> PRT
<213> pineapple <400> 16 His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gln Ile Gly Phe Pro Asp Leu Glu Ile Gln Ile His Asn Ser Trp Leu Phe Phe Pro Trp His Arg Phe Tyr Leu Tyr Phe Asn Glu Arg Ile Leu Gly Lys Leu Ile Gly Asp Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met Gln Phe Pro Ser Ile Tyr Thr Asp Pro Ser Ser Ser Leu Tyr Asp Lys Leu Arg Asp Ala Lys His Gln Pro Pro Thr Leu Ile Asp Leu Asp Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln Ile Asn His Asn Leu Ala Val Met Tyr Arg Gln Val Ile Ser Ser Gly Lys Thr Pro Glu Leu Phe Met Gly Ser Ala Tyr Arg Ala Gly Asp Gln Pro Asp Pro Gly Ala Gly Ser Val Glu Gln Lys Pro His Gly Pro Val His Val Trp Thr Gly Asp Arg Asn Gln Pro Asn Arg Glu Asp Met Gly Thr Leu Tyr Ser Ala Ala Trp Asp Pro Val Phe Phe Ala His His Gly Asn Ile Asp Arg Met Trp Tyr Val Trp Arg Asn Leu Gly Gly Lys His Arg Asn Phe Thr Asp Pro Asp Trp Leu Asn Ala Ser Phe Leu Phe Tyr Asp Glu <210> 17 <211> 1319 <212> DNA
<213> pineapple <220>
<221> CDS
<222> (1)..(1053) <400> 17 ttg ccg ttt tgg aat tgg gac gcg ccg ggg ggc atg cag atc ccg gcc 48 Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met Gln Ile Pro Ala AMENDED SHEET (Article 34) (IPEA/A~

_ CA 02290887 1999-11-18 . ' ' PCT/AU98/00362 ~:m.,:usc~.sec~-~sism ReCelveCl 19 M8y 1999 atc tac gcc gac get tcg tcc ccg ctc tac gac aag ctg cgc aat gcg 96 Ile Tyr Ala Asp Ala Ser Ser Pro Leu Tyr Asp Lys Leu Arg Asn Ala aag cac cag ccg ccg act ttg gtc gac ctc gac tac aac ggc acc gac 144 Lys His Gln Pro Pro Thr Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp ccg acc ttc acc cct gag cag cag atc gcc cac aac ctc acc atc atg 192 Pro Thr Phe Thr Pro Glu Gln Gln Ile Ala His Asn Leu Thr Ile Met tac cga cag gtg ata tcc ggc ggg aag acg ccg gag ttg ttt atg ggc 240 Tyr Arg Gln Val Ile Ser Gly Gly Lys Thr Pro Glu Leu Phe Met Gly gcg gcg tac cgc gcg ggc gac gcg cca gac ccg ggc gca ggc act cta 288 Ala Ala Tyr Arg Ala Gly Asp Ala Pro Asp Pro Gly Ala Gly Thr Leu gag ctc gtg ccg cac aac acg atg cat ttg tgg acc ggc gac ccc aac 336 Glu Leu Val Pro His Asn Thr Met His Leu Trp Thr Gly Asp Pro Asn caa ccc aac gac gaa gac atg ggc acg ttc tac gcg gcg gcg cgg gac 384 Gln Pro Asn Asp Glu Asp Met Gly Thr Phe Tyr Ala Ala Ala Arg Asp ccc atc ttc ttc gcc cac cac ggc aac gtc gac cgc atg tgg tac gtg 432 Pro Ile Phe Phe Ala His His Gly Asn Val Asp Arg Met Trp Tyr Val tgg cgg aaa ctc ggg ggc acg cac cgc gat ttc acc gac ccc gac tgg 480 Trp Arg Lys Leu Gly Gly Thr His Arg Asp Phe Thr Asp Pro Asp Trp ctc aac gcg tcc ttc ctc ttc tac gac gag aac gcg cag ctc gtc cgc 528 Leu Asn Ala Ser Phe Leu Phe Tyr Asp Glu Asn Ala Gln Leu Val Arg gtc aaa gta aag gac tgc ttg agc gcc gac gcg ctg cgg tac acg tac 576 Val Lys Val Lys Asp Cys Leu Ser Ala Asp Ala Leu Arg Tyr Thr Tyr cag gac gtc gac atc ccg tgg atc agt gcg aag ccg acg ccg aag aaa 624 Gln Asp Val Asp Ile Pro Trp Ile Ser Ala Lys Pro Thr Pro Lys Lys aca ecg ggg ggc get gcg cct tec acg aca gag get ata ttt ccg gtg 672 Thr Pro Gly Gly Ala Ala Pro Ser Thr Thr Glu Ala Ile Phe Pro Val gtg ctg gat aag ccg gtg agc tct acg gtg gcg agg ccg aag acg ggg 720 Val Leu Asp Lys Pro Val Ser Ser Thr Val Ala Arg Pro Lys Thr Gly agg agt act ggg gag gag gag gtg ttg gtg gtg gag gga atc gag ctg 768 Arg Ser Thr Gly Glu Glu Glu Val Leu Val Val Glu Gly Ile Glu Leu AMENDED SHEET (Article 34) (IPEA/ALn Received 19 May 1999 n:maast.~.seQ - txisi~

gac aag gac gtg gcc gtg aag ttc gac gtg tat ata aac gcg ccg gac 816 Asp Lys Asp Val Ala Val Lys Phe Asp Val Tyr Ile Asn Ala Pro Asp aac gaa ggg gtg ggg ccg gag gcg agc gag ttc gca ggg agc ttc gtc 864 Asn Glu Gly Val Gly Pro Glu Ala Ser Glu Phe Ala Gly Ser Phe Val cag gtg ccg cac aag cac aag aag ggg aag aag gag aag gcg agg att 912 Gln Val Pro His Lys His Lys Lys Gly Lys Lys Glu Lys Ala Arg Ile aaa acg acg ctc agg ctc ggg ata acg gac ctg ctc gag gac atc ggc 960 Lys Thr Thr Leu Arg Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly gcc gag gac gac gag agc gtg ctc gtc acg ctc gtg ccg agg ata ggc 1008 Ala Glu Asp Asp Glu Ser Val Leu Val Thr Leu Val Pro Arg Ile Gly gag ggg ttg gtc aag gtt ggt ggg cta agg atc gat ttc tcc aag 1053 Glu Gly Leu Val Lys Val Gly Gly Leu Arg Ile Asp Phe Ser Lys tgatcagcag caaattaact atacatgaaa gtaaaaaaaa ttgcatttac ctacctatag 1113 aagagaataa atgcgtatgt aatctgcccc atttgtcact tttaatttct cgagcgtgtt 1173 ctgaatgaga gttgcatgca tgcgcgcagc cataatgcct ggtatagtgt agtagtttag 1233 gcgtggatac gtataacgta cgtatgcatg tataaggaat aatgatgagt ttactatgca 1293 aaaaaaaaaa aaaaaaaaaa aaaaaa 1319 <210> 18 <211> 351 <212> PRT
<213> pineapple <400> 18 Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met Gln Ile Pro Ala Ile Tyr Ala Asp Ala Ser Ser Pro Leu Tyr Asp Lys Leu Arg Asn Ala Lys His Gln Pro Pro Thr Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp Pro Thr Phe Thr Pro Glu Gln Gln Ile Ala His Asn Leu Thr Ile Met Tyr Arg Gln Val Ile Ser Gly Gly Lys Thr Pro Glu Leu Phe Met Gly Ala Ala Tyr Arg Ala Gly Asp Ala Pro Asp Pro Gly Ala Gly Thr Leu Glu Leu Val Pro His Asn Thr Met His Leu Trp Thr Gly Asp Pro Asn AMENDED SHEET (Article 34) (IPEA/AIn ' PCT/AU98/00362 ,~,:~z~aa~sc,~.s~Q-isisw~ Received 19 May 1999 Gln Pro Asn Asp Glu Asp Met Gly Thr Phe Tyr Ala Ala Ala Arg Asp Pro Ile Phe Phe Ala His His Gly Asn Val Asp Arg Met Trp Tyr Val Tzp Arg Lys Leu Gly Gly Thr His Arg Asp Phe Thr Asp Pro Asp Trp Leu Asn Ala Ser Phe Leu Phe Tyr Asp Glu Asn Ala Gln Leu Val Arg Val Lys Val Lys Asp Cys Leu Ser Ala Asp Ala Leu Arg Tyr Thr Tyr Gln Asp Val Asp Ile Pro Trp Ile Ser Ala Lys Pro Thr Pro Lys Lys Thr Pro Gly Gly Ala Ala Pro Ser Thr Thr Glu Ala Ile Phe Pro Val Val Leu Asp Lys Pro Val Ser Ser Thr Val Ala Arg Pro Lys Thr Gly Arg Ser Thr Gly Glu Glu Glu Val Leu Val Val Glu Gly Ile Glu Leu Asp Lys Asp Val Ala Val Lys Phe Asp Val Tyr Ile Asn Ala Pro Asp Asn Glu Gly Val Gly Pro Glu Ala Ser Glu Phe Ala Gly Ser Phe Val Gln Val Pro His Lys His Lys Lys Gly Lys Lys Glu Lys Ala Arg Ile Lys Thr Thr Leu Arg Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp Asp Glu Ser Val Leu Val Thr Leu Val Pro Arg Ile Gly Glu Gly Leu Val Lys Val Gly Gly Leu Arg Ile Asp Phe Ser Lys <210> 19 <211> 2181 <212> DNA
<213> pineapple <220>
<221> CDS
<222> (2)..(1858) <400> 19 c ggt atc gat aag ctt gat cca gtg cct ggt tta ggt gta ttc act atg 49 Gly Ile Asp Lys Leu Asp Pro Val Pro Gly Leu Gly Val Phe Thr Met AMENDED SHEET (Article 34) (IPEA/ALn ' ' PCT/AU98/00362 Received 19 May 1999 .4: ;?.144366.SEQ - I S/5/99 gcc acc ctc tct aaa cta get tcc caa cca ata aca cct cca ctc tcc 97 Ala Thr Leu Ser Lys Leu Ala Ser Gln Pro Ile Thr Pro Pro Leu Ser ccg ctc cct cct ttg cat get cct tct ctc acc aaa agc ttc acc acc 145 Pro Leu Pro Pro Leu His Ala Pro Ser Leu Thr Lys Ser Phe Thr Thr acc ttc ctc tcc cct gta ggg gtc cca aac cac ccc gtc ata aga tct 193 Thr Phe Leu Ser Pro Val Gly Val Pro Asn His Pro Val Ile Arg Ser cat gca aat cta agg agc aac aag aga atg ccg aca agc ctg cgg gcc 241 His Ala Asn Leu Arg Ser Asn Lys Arg Met Pro Thr Ser Leu Arg Ala gca tcg ccc gcc gcg acc tac tcc tgg gcc ctc ggc ggg ctt tac ggt 289 Ala Ser Pro Ala Ala Thr Tyr Ser Trp Ala Leu Gly Gly Leu Tyr Gly gcc acc act ggg ctc ggc ctc aac cgt cga gcg gcc gcc gcc cct atc 337 Ala Thr Thr Gly Leu Gly Leu Asn Arg Arg Ala Ala Ala Ala Pro Ile ctg get ccc gac ctc tca act tgt ggg ccg cct gcc gac ctc cct gcc 385 Leu Ala Pro Asp Leu Ser Thr Cys Gly Pro Pro Ala Asp Leu Pro Ala tcc gcc cga ccg aca gtt tgc tgc ccg cca tac caa tcc acc atc atc 433 Ser Ala Arg Pro Thr Val Cys Cys Pro Pro Tyr Gln Ser Thr Ile Ile gac ttc aag ctc ccc ccg cga tct get ccg ctt cgc gtc cgg cct gcg 481 Asp Phe Lys Leu Pro Pro Arg Ser Ala Pro Leu Arg Val Arg Pro Ala gcc cac ttg gtt gac gcc gac tac ctg gcc aag tat aag aag gcg gtc 529 Ala His Leu Val Asp Ala Asp Tyr Leu Ala Lys Tyr Lys Lys Ala Val gag ctc atg agg gcc ctg ccg gcc gac gac ccg cgc aac ttc gta cag 577 Glu Leu Met Arg Ala Leu Pro Ala Asp Asp Pro Arg Asn Phe Val Gln caa gcg aaa gtg cac tgt gcg tat tgc gac ggc gcg tat gac caa atc 625 Gln Ala Lys Val His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gln Ile ggc ttc ccc gat ctc gag atc cag atc cac aac tcg tgg ctc ttc ttt 673 Gly Phe Pro Asp Leu Glu Ile Gln Ile His Asn Ser Trp Leu Phe Phe cct tgg cac cgg ttc tac ctc tac tcc aac gag cgc ata ctc ggg aaa 721 Pro Trp His Arg Phe Tyr Leu Tyr Ser Asn Glu Arg Ile Leu Gly Lys ctt atc ggc gac gac acg ttc gcg ctg cct ttc tgg aac tgg gac gcg 769 Leu Ile Gly Asp Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Ala ccg ggg ggc atg cag ttc ccg tct atc tac aca gac cct tca tcc tcg 817 AMENDED SHEET (Article 34) (IPEA/AIn Received 19 May 1999 .4:~2144~6f.SEQ - I~/S/99 Pro Gly Gly Met Gln Phe Pro Ser Ile Tyr Thr Asp Pro Ser Ser Ser cta tat gac aag ctg cgt gat gcg aag cac cag ccg ccg act ttg att 865 Leu Tyr Asp Lys Leu Arg Asp Ala Lys His Gln Pro Pro Thr Leu Ile gac ctc gac tac aat ggc acc gat cct acc ttc tcc cct gaa gaa cag 913 Asp Leu Asp Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln att aac cac aac ctc gcc gtc atg tac cga cag gtg ata tcc agt gga 961 Ile Asn His Asn Leu Ala Val Met Tyr Arg Gln Val Ile Ser Ser Gly aag acg cca gag ctg ttt atg ggc tca gcg tac cgc gcc ggt gac cag 1009 Lys Thr Pro Glu Leu Phe Met Gly Ser Ala Tyr Arg Ala Gly Asp Gln cct gac ccc ggc gca ggc tct gta gag cag aag ccg cac ggc ccg gtg 1057 Pro Asp Pro Gly Ala Gly Ser Val Glu Gln Lys Pro His Gly Pro Val cat gtg tgg aca ggt gat cgc aac cag ccc aat cgc gaa gac atg ggc 1105 His Val Trp Thr Gly Asp Arg Asn Gln Pro Asn Arg Glu Asp Met Gly acg ctc tac tcg gcg gcg tgg gac ccc gtc ttc ttc gca cac cac ggc 1153 Thr Leu Tyr Ser Ala Ala Trp Asp Pro Val Phe Phe Ala His His Gly aac atc gac cgc atg tgg tac gtg tgg agg aac ctt ggc ggc aag cac 1201 Asn Ile Asp Arg Met Trp Tyr Val Trp Arg Asn Leu Gly Gly Lys His cgc aac ttc acc gac ccc gac tgg ctc aac gcg tcc ttc ctg ttc tat 1249 Arg Asn Phe Thr Asp Pro Asp Trp Leu Asn Ala Ser Phe Leu Phe Tyr gat gag aat gcg cag ctc gtc cgt gtt aaa gta aaa gac tgc tta gag 1297 Asp Glu Asn Ala Gln Leu Val Arg Val Lys Val Lys Asp Cys Leu Glu gcc gac gca atg cgg tac aca tac cag gat gta gag atc ccg tgg ctc 1345 Ala Asp Ala Met Arg Tyr Thr Tyr Gln Asp Val Glu Ile Pro Trp Leu aaa gca aag ccg acg cca aag agc gcc cta cag aag ata aag agc aag 1393 Lys Ala Lys Pro Thr Pro Lys Ser Ala Leu Gln Lys Ile Lys Ser Lys gta tcg acg ctg aag gca aca cca agg ggg acg acg act acc aca gca 1441 Val Ser Thr Leu Lys Ala Thr Pro Arg Gly Thr Thr Thr Thr Thr Ala gag act aca ttt ccg gtg gtg ctg gat aag ccg gtg agt gca aca gtg 1489 Glu Thr Thr Phe Pro Val Val Leu Asp Lys Pro Val Ser Ala Thr Val get aga ccg aag gcc agg agg agt ggg aag gag aag gaa gaa gag gag 1537 Ala Arg Pro Lys Ala Arg Arg Ser Gly Lys Glu Lys Glu Glu Glu Glu AMENDED SHEET (Article 34) (1PEA/Atn ~ ~ PCT/AU98/00362 Received 19 May 1999 A .~? Li.186p.SEQ - f ~15~99 gag gtg ttg gtg gtg gag gga atc gag ttg gag aag gac gtg ttc gtg 1585 Glu Val Leu Val Val Glu Gly Ile Glu Leu Glu Lys Asp Val Phe Val aag ttt gat gtg tat ata aac tcg ccg gag cac gaa ggg gtg ggg ccg 1633 Lys Phe Asp Val Tyr Ile Asn Ser Pro Glu His Glu Gly Val Gly Pro gag gcg agt gag ttc gca ggg agc ttc gtc cac gtg cca cac aag cac 1681 Glu Ala Ser Glu Phe Ala Gly Ser Phe Val His Val Pro His Lys His aag aag gcg aag aag ggg aag gag atg gcc agg atg aac aca agg ctt 1729 Lys Lys Ala Lys Lys Gly Lys Glu Met Ala Arg Met Asn Thr Arg Leu aag ctc ggg ata acg gac ctg ctc gag gac atc ggc get gag gac gac 1777 Lys Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp Asp gag agc gtg ctc atc acg ctc gtg ccc agg agc ggc aag gga atg gtg 1825 Glu Ser Val Leu Ile Thr Leu Val Pro Arg Ser Gly Lys Gly Met Val aag gtt gga ggg cta agg att gat ttc tcc aag tgatgagcat attgtgaaga 1878 Lys Val Gly Gly Leu Arg Ile Asp Phe Ser Lys gaaaatttgc atttaccgcc ctatagaatc gaaaaattgc gtatatgtcc cattattgtt 1938 ttttttattc ttcaagcgta ttcagaataa gagttgcgtg catgcacgca tgcagccatg 1998 ttgttgtagt cgatatgtgg ggtatgtttg gatcagggat aatgatgtga actttgaatt 2058 aattattaca ctctgagaat aaattagaga gtttattatg caagttgctt ggtgtaatag 2118 atattcaaca ttgtttccta tacatctttt tttggaagaa aaaaaaaaaa aaaaaaaatc 2178 gat 2181 <210> 20 <211> 619 <212> PRT

<213> pineapple <400> 20 Gly Ile LysLeu Asp ValProGly LeuGly ValPheThr Met Asp Pro Ala Thr SerLys Leu SerGlnPro IleThr ProProLeu Ser Leu Ala Pro Leu ProLeu His ProSerLeu ThrLys SerPheThr Thr Pro Ala Thr Phe SerPro Val ValProAsn HisPro ValIleArg Ser Leu Gly AMENDED SHEET (Article 34) (IPEA/Ain CA 02290887 1999-m-is PCT/AU98/00362 Received 19 May 1999 , .~ ~:~iasac~.sFQ- ix,~sW

His Ala Asn Leu Arg Ser Asn Lys F,rg Met Pro Thr Ser Leu Arg Ala Ala Ser Pro Ala Ala Thr Tyr Ser Trp Ala Leu Gly Gly Leu Tyr Gly Ala Thr Thr Gly Leu Gly Leu Asn Arg Arg Ala Ala Ala Ala Pro Ile Leu Ala Pro Asp Leu Ser Thr Cys Gly Pro Pro Ala Asp Leu Pro Ala Ser Ala Arg Pro Thr Val Cys Cys Pro Pro Tyr Gln Ser Thr Ile Ile Asp Phe Lys Leu Pro Pro Arg Ser Ala Pro Leu Arg Val Arg Pro Ala Ala His Leu Val Asp Ala Asp Tyr Leu Ala Lys Tyr Lys Lys Ala Val Glu Leu Met Arg Ala Leu Pro Ala Asp Asp Pro Arg Asn Phe Val Gln Gln Ala Lys Val His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gln Ile Gly Phe Pro Asp Leu Glu Ile Gln Ile His Asn Ser Trp Leu Phe Phe Pro Trp His Arg Phe Tyr Leu Tyr Ser Asn Glu Arg Ile Leu Gly Lys Leu Ile Gly Asp Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met Gln Phe Pro Ser Ile Tyr Thr Asp Pro Ser Ser Ser Leu Tyr Asp Lys Leu Arg Asp Ala Lys His Gln Pro Pro Thr Leu Ile Asp Leu Asp Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln Ile Asn His Asn Leu Ala Val Met Tyr Arg Gln Val Ile Ser Ser Gly Lys Thr Pro Glu Leu Phe Met Gly Ser Ala Tyr Arg Ala Gly Asp Gln Pro Asp Pro Gly Ala Gly Ser Val Glu Gln Lys Pro His Gly Pro Val His Val Trp Thr Gly Asp Arg Asn Gln Pro Asn Arg Glu Asp Met Gly Thr Leu Tyr Ser Ala Ala Trp Asp Pro Val Phe Phe Ala His His Gly Asn Ile Asp Arg Met Trp Tyr Val Trp Arg Asn Leu Gly Gly Lys His AMENDED SHEET (Article 34) (IPEA/ALn CA 02290887 1999-m-is PCT/AU98/00362 ~ ~ Received 19 May 1999 .. A:~IJJSfiC.SEQ- 1815/99 Arg Asn Phe Thr Asp Pro Asp Trp Leu Asn Ala Ser Phe Leu Phe Tyr Asp Glu Asn Ala Gln Leu Val Arg Val Lys Val Lys Asp Cys Leu Glu Ala Asp Ala Met Arg Tyr Thr Tyr Gln Asp Val Glu Ile Pro Trp Leu Lys Ala Lys Pro Thr Pro Lys Ser Ala Leu Gln Lys Ile Lys Ser Lys Val Ser Thr Leu Lys Ala Thr Pro Arg Gly Thr Thr Thr Thr Thr Ala Glu Thr Thr Phe Pro Val Val Leu Asp Lys Pro Val Ser Ala Thr Val Ala Arg Pro Lys Ala Arg Arg Ser Gly Lys Glu Lys Glu Glu Glu Glu Glu Val Leu Val Val Glu Gly Ile Glu Leu Glu Lys Asp Val Phe Val Lys Phe Asp Val Tyr Ile Asn Ser Pro Glu His Glu Gly Val Gly Pro Glu Ala Ser Glu Phe Ala Gly Ser Phe Val His Val Pro His Lys His Lys Lys Ala Lys Lys Gly Lys Glu Met Ala Arg Met Asn Thr Arg Leu Lys Leu Gly Ile Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp Asp Glu Ser Val Leu Ile Thr Leu Val Pro Arg Ser Gly Lys Gly Met Val Lys Val Gly Gly Leu Arg Ile Asp Phe Ser Lys <210> 21 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 21 gcctgcagtt ytcrtcrtag as 22 <210> 22 <211> 28 <212> DNA
<213> Artificial Sequence AMENDED SHEET (Article 34) (IPEA/AU) ~' ~ ~ Received 19 May 1999 A:~~14t8C~C.SEQ- IB/5/99 <220>
<223> Description of Artificial Sequence: primer <400> 22 gcgaattcga tccnacntty gckttncc 28 <210> 23 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 23 gcgaattctn caytgygcnt aytg 24 <210> 24 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 24 gcgaattctt nccntwytgg aaytggg 27 <210> 25 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 25 gcctgcagcc acatnckrtc nacrtt 26 <210> 26 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 26 gcctgcagtt ytcrtcrtag as 22 <210> 27 <211> 25 <212 > DNA
<213> Artificial Sequence AMENDED SHEET (Article 34) (IPEA/Ai~

CA 02290887 1999-m-is PCT/AU98/00362 .
Received 19 May 1999 A:'~~.Iqq8fit.SEQ - IR/5/99 <220>
<223> Description of Artificial Sequence: primer <400> 27 gttgctcttc ttaggctcgg cttac 25 <210> 28 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 28 gactcgagtc gacatcga 18 <210> 29 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 29 atatcacctg tcggtacatg acggc 25 <210> 30 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 30 gtgccattgt agtcgaggtc aatca 25 <210> 31 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 31 ccagtgcctg gtttaggtgt attcac 26 <210> 32 <211> 35 <212> DNA
<213> Artificial Sequence <220>
AMENDED SHEET (Article 34) (IPEA/Ain ' ~ ~. , " - PCT/AU98/00362 .A: Ziaasc~.ssc~- is~s;99 ReCelvec~ 19 May 1999 <223> Description of Artificial ~°quence: primer <400> 32 gactcgagtc gacatcgatt tttttttttt ttttt 35 <210> 33 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 33 gactcgagtc gacatcga 18 AMENDED SHEET (Article 34) (IPEA/Ain

Claims

WE CLAIM:

1. An isolated nucleic acid molecule that comprises a nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a PPO polypeptide of banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ
ID NOS:
2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 or comprising the copper-binding site of any one of said amino acid sequences.

2. An isolated nucleic acid molecule that encodes a PPO polypeptide of banana, tobacco or pineapple wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19;
(ii) a fragment of (i) comprising a nucleotide sequence that encodes the copper-binding site of a PPO polypeptide;
(iii) a degenerate nucleotide sequence of (i) or (ii); and (iv) a nucleotide sequence that is complementary to (i) or (ii) or (iii).

3. The isolated nucleotide sequence according to claims 1 or 2 wherein the copper-binding site is the CuA binding site of said banana, tobacco or pineapple PPO polypeptide.

4. The isolated nucleotide sequence according to claims 1 or 2 wherein the copper-binding site is the CuB binding site of said banana, tobacco or pineapple PPO polypeptide.

5. The isolated nucleic acid molecule according to any one of claims 1 to 4, wherein the PPO polypeptide of banana is at least expressed in banana peel.

6. The isolated nucleic acid molecule according to any one of claims 1 to 4, wherein the PPO polypeptide of tobacco is at least expressed in tobacco leaves.

7. The isolated nucleic acid molecule according to any one of claims 1 to 4, wherein the PPO polypeptide of pineapple is at least expressed in pineapple fruit.

8. A recombinant vector comprising the isolated nucleic acid molecule according to any one of claims 1 to 7 inserted within a vector molecule.

9. The recombinant vector according to claim 8 wherein the vector is a plasmid expression vector.

10. The recombinant vector according to claim 9 wherein the plasmid expression vector is Bluescript SK+.

11. The recombinant vector according to claim 8 wherein the vector is a binary vector suitable for introducing into a plant cell, tissue or organ.

12. The recombinant vector according to any one of claims 8 to 11 wherein the vector is capable of being replicated and the PPO-encoding nucleotide sequence is capable of being transcribed and translated in a unicellular organism or in a plant.

13. A method of increasing the level of banana, pineapple or tobacco PPO
activity in a plant or a cell, tissue or organ thereof, said method comprising:
(i) introducing a nucleotide sequence to said plant or a cell, tissue or organ thereof which sequence encodes a PPO polypeptide of banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 or an enzymatically-active PPO polypeptide comprising the copper-binding site of any one of said amino acid sequences; and (ii) expressing said nucleotide sequence to produce an enzymatically-active PPO
polypeptide.

14. A method of increasing the level of banana, pineapple or tobacco PPO
activity in a plant or a cell, tissue or organ thereof, said method comprising:
(i) introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 or a degenerate nucleotide sequence thereof; and (ii) expressing said nucleic acid molecule to produce an enzymatically-active PPO
polypeptide.

15. A method of decreasing the level of PPO activity in a plant or a cell, tissue or organ thereof, said method comprising introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which comprises a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence which encodes a PPO polypeptide of banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS:
2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 or the copper-binding site of any one of said amino acid sequences;
(ii) a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19;
(iii) a fragment of (ii) comprising a nucleotide sequence that encodes the copper-binding site of a PPO polypeptide; and (iv) a nucleotide sequence that is complementary to (i) or (ii) or (iii).

16. The method according to claim 15 further comprising expressing the introduced nucleic acid molecule to produce sense or antisense RNA therefrom.

17. The method according to claims 15 or 16 wherein the PPO activity is decreased by co-suppression of the endogenous PPO-encoding genes that would otherwise be expressed in the plant or a cell, tissue or organ thereof.

18. The method according to claims 15 or 16, wherein the PPO activity is decreased by the expression of antisense RNA that is complementary to RNA encoded by an endogenous PPO-encoding gene that would otherwise be expressed in the plant or a cell, tissue or organ thereof.

19. The method according to any one of claims 13 to 18 wherein the nucleic acid molecule is introduced into the plant or a cell, tissue or organ thereof by means of Agrobacterium-mediated transformation.

20. The method according to any one of claims 13 to 18 wherein the nucleic acid molecule is introduced into the plant or a cell, tissue or organ thereof by means of microparticle bombardment using a nucleic acid-coated microprojectile.

21. A transformed plant comprising the isolated nucleic acid molecule according to any one of claims 1 to 7 or a plant part, progeny or propagule thereof that also comprises said nucleic acid molecule.

22. A transformed plant comprising the recombinant vector according to any one of claims 8, 9, 11 or 12 or a plant part, progeny or propagule thereof that also comprises said nucleic acid molecule.