MXPA99010707A - Polyphenol oxidase genes from banana, tobacco and pineapple - Google Patents

Abstract

La presente invención proporciona métodos para prepararácidos nucleicos que codifican la polifenoloxidasa (PPO), fragmentos y derivados de la misma. La presente invención también proporcionaácidos nucleicos que codifican la PPO de plátano, tabaco o piña o el antisentido para la PPO de plátano, tabaco o piña, fragmentos y derivados de la misma. También se proporcionan los vectores que incluyen talesácidos nucleicos, los métodos para utilizar talesácidos nucleicos y las plantas transgénicas.

Description

b POLYPHENOL OXIDASE GENES FROM BANANA, TOBACCO AND PIÑA Description of the Invention The present invention relates to the isolation of genes encoding polyphenol oxidase (PPO) from plants. The browning of plant tissues occurs frequently following the injury or damage, and this usually results in a deterioration of the fruits and vegetables. Undesirable browning also occurs during the processing of plant materials to produce food or other products. Measures are taken during transport, storage and processing to prevent these browning actions. Frequently this involves the use of chemical compounds such as sulfur dioxide, but the use of these substances is likely to be restricted in the future due to concerns about their safety and safety. acceptance by the consumer. For example, the US Food and Drug Administration banned the use of sulfite for most fresh fruits and vegetables in 1986. the production of fruit and vegetable varieties with a susceptibility inherently low to browning would eliminate the need for these chemical treatments. It will be understood that browning in plants is predominantly catalyzed by the PPO enzyme. The PPO is located in the plastids of the plant cells, while the phenolic substrates of the enzyme are stored in the vacuoles of the plant cell. This division into compartments prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed. The prior art includes the International Application PCT / AU92 / 00356 given to the present applicant, which describes the cloning of PPO genes from grapevines, potato leaves, apples and potato tubers. This application recognizes that concentrations of PPO in plants can be manipulated by increasing or decreasing the expression of the PPO gene. The application also identifies two copper binding sites conserved in PPO genes, designated CuA and CuB. However, the method described in PCT / AU92 / 00356, which was used to clone the PPO genes from apple and potato involved the use of an oligo dT reverse primer for the polymerase chain reaction (PCR). While the method is acceptable, in some tissues, it does not cause a strong band in the predicted size, or otherwise, it causes 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, the method includes providing a source of a polypeptide having PPO activity, a first initiator having a sequence that corresponds to a conserved region of PPO, and a second initiator having a sequence corresponding to a second conserved region of orientation of PPO; isolate the RNA from the source of the polypeptide having PPO activity; treat RNA to build copy DNA (cDNA) from it, - and amplify the cDNA formed in this manner using the first and second primers. The Applicant has found that the method of the present invention, which involves the use of a second PPO-based primer, means that there is a lower probability that other genes (which are not PPO) are amplified. Additionally, the method of the present invention dramatically increases the amount of genuine product formed in most cases. On the other hand, the aggregate specificity provided by the second PPO-based initiator makes it possible to clone PPO more easily from certain plants in which it was difficult to obtain a clone using an initiator 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 and fragments and derivatives thereof, the method includes rooting a source of a polypeptide having banana PPO activity , tobacco or pineapple, a first initiator who has a sequence corresponding to a first conserved region of PPO of banana, tobacco or pineapple, and a second initiator having a sequence corresponding to a second conserved region of PPO of banana, tobacco or pineapple; Isolate RNA from the source of polypeptide having banana, tobacco or pineapple PPO activity; treat the RNA to build DNA copy (cDNA) from it; and amplifying the cDNA formed in this manner 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. For example, these terms include sequences that differ from the specific sequences given in the examples herein but which, due to the degeneracy of the genetic code, encode the same protein. Applicants have found that PPO families exist in most plants. Thus, it is likely that there are other PPO genes in banana, tobacco and / or pineapple, in addition to those that are isolated. These could be cloned using the methods of the present invention. Thus, the terms "nucleic acid encoding banana / tobacco / pineapple PPO" and "plantain / tobacco / pineapple PPO gene" should be understood to include banana / tobacco / pineapple PPP genes different from those specific genes that They have been isolated. The terms may also include presequences such as the chloroplast transit sequence as well as sequences encoding mature PPO protein. The term "derivative" as used herein includes nucleic acids that have been chemically modified or otherwise modified, for example mutated or labeled, or nucleic acids that incorporate a catalytic hydrolysis site. The term "fragment" includes functionally active fragments of a PPO gene that are capable of altering the expression of the PPO genes. Examples of gene alteration may include up-regulation or sub-regulation of the gene, gene coding, gene transcription, gene binding or sequence stability of the gene. The source of the polypeptide having PPO activity is preferably a source of polypeptide having PPO activity of banana or tobacco or pineapple. The source in the polypeptide having PPO activity may be the banana peel, preferably immature banana peel. More preferably the skin of the fruit of the immature banana. The source of the polypeptide having tobacco PPO activity may be tobacco leaves, preferably immature tobacco leaves. The source of the polypeptide having pineapple PPO activity may be the fruit of the pineapple, preferably the fruit pulp of the pineapple, more preferably the pineapple fruit pulp which exhibits a brown heart disorder. The RNA can be isolated by any suitable method including extraction for example with a detergent such as C , the use of a notched oligo-dT column as described in PCT / AU92 / 00356 and PCT / AU96 / 00310, the full description of each application is incorporated herein by reference, by the use of a commercially available case such as the system PolyATratct 1000 from Promega Corporation. The step of treating RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reversed transcriptase and an adapter initiator to form cAD. The adapter initiator can be an oligonucleotide adapter primer that includes the following sequence or part of 1-a itself: '-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3' The step of treating RNA to construct cDNA according to this aspect of the present invention may include treating the RNA against reverse transcriptase and a reverse initiator to form cDNA. The adapter initiator can be replaced with a reverse primer having a sequence corresponding to a conserved region of PPO genes that include the following sequence or part thereof: REV2: 5 '-GCCTGCAGT [TC] TC [AG] TAGAA-3 The first primer has a sequence corresponding to a first conserved region of PPO. Preferably the first initiator 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 initiator 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 initiators can be degenerate. The first initiator may include one of the following sequences or part thereof: GEN8: 5 '- GCGAATTCGATCCIACITT [TC] GC [GT] TTICC-3'. GEN9: 5 'GCGAATTCTLCA [TC] TG [TC] GCITA [TC] TG-3'. GENIO: 5 'GCGAATTCTTCCI [TA] [TC] TGGA [TC] TGGG- 3' The second initiator may include the following sequences or part thereof REVI: 5 '-GCCTGCAGCCACATIC [TG] [AG] TCIAC [AG] TT-3'. REV2: 5 'GCCTGCAGTT [TC] TC [AG] TC [AG] TAGAA-3'.

The cDNA can be amplified using the polymerase chain reaction (PCR). Those skilled in the art will appreciate that if the CU binding sites are internal, the isolated nucleic acid will be a fragment of the PPO gene that lacks the 3 'and 5' terms. However, it is possible to determine the complete nucleic acid sequence of the PPO gene and prepare or isolate nucleic acid encoding such a PPO or antisense to such a 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, the method includes providing a source of polypeptide having PPO activity an initiator in direct orientation; Y an adapter initiator; isolate RNA from the source of polypeptide having PPO activity; treat the RNA to build cDNA from it; and amplify the cDNA formed in this manner 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, the method includes providing a source of polypeptide having PPO activity, an anchor; initiators in antisense orientation; and an anchor primer, - isolate RNA from the source of polypeptide having PPO activity; treat the RNA to build cDNA from it; joining the anchor to the 5 'end of the cDNA formed in this manner; and amplify the cDNA using the primers.

The source of polypeptide having PPO activity is preferably a source of polypeptide having PPO activity of banana or tobacco or pineapple. The source of the polypeptide having banana PPO activity may be the banana peel, preferably the peel of the immature banana. More preferably the skin of the fruit of the immature banana. The source of the polypeptide having tobacco PPO activity may be tobacco leaves, preferably immature tobacco leaves. The source of the polypeptide having pineapple PPO activity may be pineapple fruit, preferably pineapple fruit pulp, more preferably pineapple fruit pulp exhibiting brown heart disorder. The RNA can be isolated by any suitable method, including extraction, for example with a detergent such as C , the use of a notched oligo-dT column as described in PCT / AU92 / 00356 or PCT / AU96 / 00310, the description The completeness of each patent application is incorporated herein by reference, or the use of a commercially available kit such as the PolyATtract 1000 system from Promega Corporation. The stage of treating RNA to build cDNA according to this aspect of the present invention may include treating RNA with reverse transcriptase and an adapter primer to form cDNA. The adapter initiator can be an oligonucleotide adapter primer that includes one or both of the following sequences: r -GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3 ' The adapter initiator can be replaced with a re? Exs_a_ primer having a sequence corresponding to a conserved region of PPO genes that includes the following sequence or part thereof: REV2: 5 '-GCCTGCAGTT [TC] TC [AG] TC [AG] TAGAA-3' The initiator in direct orientation can be a specific PPO initiator of banana, tobacco or pineapple. The adapter initiator may include the following sequence or part thereof: '-GACTCGAGTCGACATCGA-3.

The primers in antisense orientation can be specific PPO initiators of banana, tobacco or pineapple. The anchor can be of any suitable type. The anchor can be linked by linker for example using T4 RNA ligase. The anchor initiator may be capable of hybridizing with the anchor. The cDNA can be amplified using PCR. Those skilled in the art will appreciate that using the methods of the present invention it is possible to determine for the complete nucleic acid sequence of the PPO of interest and to prepare or isolate nucleic acid encoding such a PPO or antisense to such a PPO. In a further aspect of the present invention, a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof is provided. Preferably the nucleic acid has the sequence shown in Figures 1, 2, 3 or 4, fragments and derivatives thereof, and substantially homologous sequences.

In a further aspect, the present invention provides a nucleic acid encoding tobacco or anti-sense PPO to the PPO of tobacco, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figures 5, 6 or 7, fragments and derivatives thereof, and substantially homologous sequences. In a further aspect of the present invention, a nucleic acid encoding pineapple or antisense PPO is provided to the pineapple PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Figures 8, 9 or 10, fragments and derivatives thereof, and sub sequences and homologous sub sequences. The nucleic acid can be prepared by a method described above. The nucleic acid can be modified as for example, by inclusion of a catalytic hydrolysis site. In a further aspect of the present invention there is provided a method for preparing a recombinant vector that includes a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof, The method includes providing nucleic acid encoding plantain PPO or antisense to the banana PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to display the nucleic acid within the vector. In a further aspect of the present invention there is provided a method for preparing a recombinant vector that includes a nucleic acid that encodes tobacco PPO or antisense to the PPO of tobacco, fragments and derivatives thereof, the method includes providing nucleic acid that encodes tobacco or antisense PPO to the tobacco PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to display the nucleic acid within the vector.

In a further aspect of the present _ invention provides a method for preparing a recombinant vector that includes a pineapple or antisense PPO-encoding nucleic acid to pineapple PPO, fragments and derivatives thereof, the method includes providing nucleic acid encoding pineapple or antisense PPO to the Pineapple PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and 1 vector to display the nucleic acid within the vector. The nucleic acid can be prepared by a method described above. The nucleic acid can be modified, for example by inclusion of a catalytic hydrolysis site. The vector can be a plasmid expression vector. For example it has been found that Bluescript SK * is suitable. Alternatively, the vector can be a binary vector. The recombinant vector may contain a promoter, preferably a constitutive promoter upstream of the nucleic acid.

The cloning step can take any suitable form. A preferred form may include fractionating the cDNA, for example on a column or a gel; isolating the fragment of the expected size, for example from the column or gel; and ligating the fragment into a suitable restriction enzyme site of the vector, for example the EcoR site of a Bluescript SK * vector. To test the clones formed in this way, a suitable microorganism can be transformed with the vector, the cultured microorganism and the polypeptide encoded therein. The microorganism can be a strain of Escherichia coli, for example E. DH5 coli has been found to be suitable. Alternatively, appropriate vectors can be used to transform plants. In a further aspect of the present invention there is provided a recombinant vector that includes a nucleic acid that encodes plantain PPO or antisense to the banana PPO, fragments and derivatives thereof, the vector is capable of being replicated, transcribed and transladated into a single cell organism, or alternatively in a plant . In a further aspect of the present invention there is provided a recombinant vector that includes a nucleic acid that encodes tobacco PPO or antisense to the PPO of tobacco, fragments and derivatives thereof, the vector is capable of being replicated, transcribed and translated into a unicellular organism or alternatively in a plant. In a further aspect of the present invention there is provided a recombinant vector that includes a pineapple PPO or antisense PPO-encoding nucleic acid to the pineapple PPO, fragments and derivatives thereof, the vector is capable of being replicated, transcribed or translated into a unicellular organism or alternatively in a plant. The nucleic acid can be prepared by a method described above. The nucleic acid can be modified, for example by inclusion of a catalytic hydrolysis site. The vector can be a plasmid expression vector. For example it has been found that Bluescript SK * is suitable. Alternatively, the vector can be a binary vector. The recombinant vector may contain __ a promoter, preferably a constitutive promoter upstream of the nucleic acid encoding the banana, tobacco or pineapple PPO or antisense to the banana, tobacco or pineapple PPO, fragments and derivatives thereof. The microorganism can be a strain of Escherichia coli, for example E. col i DH5 has been found to be adequate. In a further aspect of the present invention there is provided a method for lowering the level of PPO- activity in a plant tissue, the method includes providing a nucleic acid encoding banana PPO, a modified nucleic acid "which" encodes PPO of plantain, a modified nucleic acid encoding plantain PPO, or an antisense nucleic acid to banana PPO, fragments and derivatives thereof; and a plant sample; and introducing the nucleic acid into the plant sample to produce a transgenic plant. In a further aspect of the present invention there is provided a method for decreasing the level of PPO activity in a plant tissue, the method includes providing a nucleic acid encoding tobacco PPO, a modified nucleic acid encoding tobacco PPO, or a nucleic acid amphisense to tobacco PFO, fragments and derivatives thereof; and a plant sample; and introducing the nucleic acid into the plant sample to produce a transgenic plant. In a further aspect of the present invention there is provided a method for lowering the level of PPO activity in a plant tissue, the method includes providing a nucleic acid encoding pineapple PPO, a modified nucleic acid encoding pineapple PPO or a nucleic acid antisense to the pineapple PPO, fragments and derivatives thereof; and a plant sample; and introducing the nucleic acid into the plant sample to produce a transgenic plant. The activity of the PPO can be decreased by the use of direct constructs (cosupresión). Alternatively, the nucleic acid may include a sequence encoding banana or tobacco or pineapple antisense mRNA to 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 hydrolysis site (ribozyme). The nucleic acid can be included in a recombinant vector as described above. In a preferred aspect, the nucleic acid can be included in a binary vector. In a further preferred aspect, the introduction of a binary vector into the plant can be by infection of the plant with an Agrobacterium containing the binary vector, or by bombardment with microprojectiles coated with nucleic acid. 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., Bio / tecnology (1995) 13: 466-492; Michelmore et al., Plant Cell Reports (1987) 6: 439-442; and Curtis et al. ^ Journal of Experimental Botany (1994) 45: 1141-1149, the full descriptions of each are incorporated herein by reference. Methods for transforming banana, tobacco or pineapple by bombardment with microprojectiles coated with DNA are known to those skilled in the art, and are described in, for example, Sagi et al., Bio / technology (1995) 13: 481-485, full description of which is incorporated herein by reference. In a further aspect of the present invention, there is provided a method for increasing the level of activity of the PPO in the tissue of a plant, the method includes: providing a nucleic acid encoding banana PPO, or a fragment thereof; and a vegetable sample; and introducing the nucleic acid into the plant sample, to produce a transgenic plant. In a further aspect of the present invention, there is provided a method for increasing the level of PPO activity in the tissue of a plant, the method includes: providing - - - a nucleic acid encoding tobacco PPO, or a fragment of the same; Y a vegetable sample; and introducing the nucleic acid into the plant sample, to produce a transgenic plant. In a further aspect of the present invention, there is provided a method for increasing the level of PPO activity in the tissue of a plant, the method includes: providing a nucleic acid encoding pineapple PPO, or a fragment thereof; and a vegetable sample; and introducing the nucleic acid into the plant sample, to produce a transgenic plant. The nucleic acid can be included in a recombinant vector as described above. In a preferred aspect, the nucleic acid can be included in a binary vector. In a further preferred aspect, the introduction of the binary vector into the plant can be by infection of the plant with an Agrobacterium containing the primary vector, or by bombardment with microprojectiles coated with nucleic acid. The plant can be of any suitable type. However, the method is particularly applicable to banana, tobacco or pineapple.

In a further aspect of the present invention a transgenic plant is provided, the plant contains nucleic acid capable of modifying expression of the normal banana PPO gene. _ The plant can be of any suitable type. Preferably, the plant is banana. In a further aspect of the present invention a transgenic plant is provided, the plant contains nucleic acid capable of modifying expression of the normal tobacco PPO gene. The plant can be of any suitable type. Preferably, the plant is tobacco. In a further aspect of the present invention a transgenic plant is provided, the plant contains nucleic acid capable of modifying expression of the normal pineapple PPO gene. The plant can be of any suitable type. Preferably, the plant is pineapple. The nucleic acid can be as described above. In a still further aspect of the present invention there is provided a plant vaccine which includes 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 vaccine which includes nucleic acid encoding tobacco PPO, or antisense to the PPO of tobacco, fragments and derivatives thereof. In a still further aspect of the present invention there is provided a plant vaccine which includes nucleic acid encoding pineapple PPO, or antisense to pineapple PPO, fragments and derivatives thereof. The present invention will now be described more fully with reference to the "appended Examples." It should be understood, however, that the following description is illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above. FIGURES: FIGURE 1: The nucleotide sequence of the BPP02 cDNA (SEQ ID NO: 1) and derived protein sequence (SEQ ID NO: 2) encoding part of a banana PPO protein FIGURE 2: The nucleotide sequence of the BPP08 cDNA (SEQ ID NO: 3) and derived protein sequence (SEQ ID NO: 4) that encodes part of a PPO protein - eleven of banana. FIGURE 3: The nucleotide sequence of the BANPP034 cDNA (SEQ ID NO: 5) and the derived protein sequence (SEQ ID NO: 6) encoding part of a banana PPO protein. FIGURE 4: The nucleotide sequence "of the BPP035 cDNA (SEQ ID NO: 7) and derived protein sequence (SEQ ID NO: 8) encoding part of a banana PPO protein FIGURE 5: The nucleotide sequence of the cDNA TOBPP06 (SEQ ID NO: 9) and derived protein sequence (SEQ ID NO: 10) encoding part of a banana PPO protein FIGURE 6: The nucleotide sequence of the TOBPP025 cDNA (SEQ ID NO: ll) and sequence of derived protein (SEQ ID NO: 12) encoding part of a tobacco PPO protein FIGURE 7: The nucleotide sequence of the TOBPP026 cDNA (SEQ ID NO: 13) and derived protein sequence (SEQ ID NO: 14) encoding part of a tobacco PPO protein FIGURE 8: The nucleotide sequence of the PINPPO20 cDNA (SEQ ID NO: 15) and derived protein sequence (SEQ ID NO: 16) which encodes part of a pineapple PPO protein .

FIGURE 9: The nucleotide sequence of the PINPP02 cDNA (SEQ ID NO: 17) and derived protein sequence (SEQ ID NO: 18) which encodes part of a pineapple PPO protein. FIGURE 10: The nucleotide sequence of the PINPPOFL cDNA (SEQ ID NO: 19) and derived protein sequence (SEQ ID NO: 20) that encodes part of a pineapple PPO protein. EXAMPLE 1: Cloning of Banana Cascara PPO Genes Total RNA was isolated from the skin of immature banana fruit. Fruit tissue (3 g) was frozen, and ground to a fine powder in liquid nitrogen with a coffee mill, then added to 20 ml of extraction buffer (2% hexadecyl-1-trimethylammonium bromide (C ). , 2% polyvinyl pyrrolidone, 100 mM Tris-HCl, pH 8.0, 25 mM EDTA, 2 M NaCl, 0.05% spermidine, 2% β-mercaptoretanol) at 65 ° C. The extract was mixed with 20 ml of chloroform / IAA, then subjected to centrifugation for 20 minutes at 5,000 RPM, and the aqueous phase was re-extracted with chloroform / lAA. The aqueous phase was filtered through Miracloth, and 0.25 volumes of 10 M LiCl were added, then the sample was incubated overnight at 4 ° C before undergo centrifugation for 20 minutes at 8,000 RPM. The supernatant was removed, and the pellet was resuspended in 0.5 ml of 1 M NaCl, 0.5% SDS, 10 M Tris, pH 8.0, 1 mM EDTA. The RNA was extracted once with an equal volume of chloroform / IAA, and 2 volumes of ethanol were added. After incubation for 40 minutes at -70 ° C, the solution was subjected to centrifugation 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 μl of sterile water. The first strand of cDNA was synthesized from 10 μg of total RNA _ with reverse transcriptase as described in Reference 2, using an oligosaccharide initiator adapter -dT (Reference 1): B26 (SEQ ID NO: 32): (5) '-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3'). Oligonucleotide primers were designed based on known plant PPO DNA sequences. The comparison of a number of PPO sequences from a range of different plants allowed the identification of the conserved regions of the gene, which are mainly in or near the regions that encode the two copper binding sites, CuA and CuB (2). Forward primers designed around the CuA site (GEN8, GEN9 and GEN 10) and reverse primers designed around the site were synthesized CuB (REVI and REV2): GEN8 (SEQ ID NO: 22): (5 '-GCGAATTCGATCCIACITT [TC] GC [GT] TTICC- 3') GEN9 (SEQ ID NO: 23): (5 '-GCGAATTCTICA [TC] TG [TC] GCITA [TC] TG-3 ') GENIO (SEQ ID NO: 24): (5 '-GCGAATTCTTICCIT [TA] [TC] TGGAA [TC] - TGGG-3') REVI (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 reaction of the first strand was amplified by the polymerase chain reaction (PCR) essentially according to the Frohman method (1), using GEN primers and ~ REV, each in one final concentration of 1 μM (2). Amplification involved an initial program of 2 cycles of denaturation at 94 ° C for 1 minute, annealing at 37 ° C for 2 minutes, a slow slope at 72 ° C for 3 minutes, followed by 33 cycles of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and elongation at 72 ° C for 3 minutes minutes 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 agarose gel, and the bands of interest were cut. The DNA was purified from the agarose with a QIAquic PCR Purification kit (Qiagen). The purified DNA was cloned into the Bluescript SK * vector cut with Eco-RV (Stratagene) to which T was placed at the tail end with Taq Polymerase, and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones that had an insert of the predicted size were selected and their DNA sequence was determined by determination of the automated sequence. Two clones of putative banana PPO (BPP02 and BPP08) were identified, based on their homology to known plant PPO genes. The 3 'end of BPP02 was cloned using an initiator designed to the BPP02 sequence: BAN8F (SEQ ID NO: 27): (5'-GTTGCTCTTCTTAGGCTCGGCTTAC-3') at a final concentration of 1 μM, and an adapter initiator of B25 : B25: (SEQ ID NO: 28): (5 '-GACTCGAGTCGACATCGA-3') in a final concentration of 1 μM (reference 1).

The amplification involved 35 cycles of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and elongation at 72 ° C for 3 minutes. 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 agarose gel, and the bands of interest were cut. The DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen). The purified DNA was cloned into the Bluescript SK * vector cut with Eco-RV (Stratagene) to which T was placed at the tail end with Taq Polymerase, and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones that had an insert of the predicted size (1150 bp) were selected and their DNA sequence was determined by automated sequence determination. Two putative plantain PPO clones (BANPP034 and BANPP035) were identified, based on their homology to known plant PPO genes. The sequence of BANPP034 was identical to that of BPP02. E EMP O 2: Cloning of Tobacco Leaf PPO genes Total RNA from immature leaves (1-3 cm long) was isolated from plants grown in the greenhouse. Approximately 2 g of frozen leaf material were ground to a fine powder in liquid nitrogen, then extracted in 15 ml of extraction buffer (50 mM Tris-HCl, pH 9.0, 150 mM LiCl, 5 mM EDTA, 5% SDS and 0.6% ß -mercaptoethanol) by shaking vigorously in a tube with a 50 ml screw cap for 1-2 minutes. Approximately 15 ml of phenol / chloroform / IAA (25: 24: 1) was added, and the homogenate was mixed and then subjected to centrifugation for 15 minutes at 5,000 RPM and 4 ° C. The upper aqueous phase was removed, and re-extracted twice with phenol / chloro ormo / IAA and then once with chloroform / IAA and then subjected to centrifugation for 10 minutes at 5,000 RPM and 4 ° C. The supernatant was removed, LiCl was added to a final concentration of 2 M, and the mixture was incubated overnight at 4 ° C. centrifugation for 10 minutes at 8,000 RPM and 4 ° C the supernatant was removed, and the pellet was resuspended in 6 ml of 0.4 M LiCl, then 2 ml of 8 M LiCl was added and the mixture was incubated overnight 4 ° C. The mixture was subjected to centrifugation for 10 minutes at 8,000 RPM and 4 ° C, the supernatant was removed, and the pellet was resuspended in 0.5 ml of sterile water, and subjected to centrifugation briefly, to remove any insoluble material. MRNA was isolated from total RNA using a PolyATtract kit (Promega). First-strand cDNA was synthesized from 10 μg of total RNA or 2 μg of reverse transcriptase mRNA as described in Reference 2, using an oligo-dT primer adapter (Reference 1): B26: (5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT -3 ') The reaction of the first strand was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman (1) using primers GEN and REV described in Example 1, each in a final concentration of 1 μM (2). Amplification involved an initial program of 2 cycles of denaturation at 94 ° C for 1 minute, annealing at 37 ° C for 2 minutes, a slow slope at 72 ° C for 2 minutes, and elongation at 72 ° C for 3 minutes, followed for 28 cycles of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and elongation at 72 ° C for 3 minutes. A sample of the amplified DNA was run on an agarose gel and stained with bromide of ethidium, to determine the size of the PCR products. The remainder was run on a low melting agarose gel, and the bands of interest were cut. The DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen). The purified DNA was cloned into a Bluescript SK * vector cut with Eco-RV (Stratagene) to which T was placed at the tail end with Taq Polymerase, and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones that had an insert of the predicted size were selected and their DNA sequence was determined by automated sequence determination. Three putative tobacco PPO clones (TOBPP06, TOBPP025 and TOBPP026) were identified, based on their homology to known plant PPO genes. EXAMPLE 3: Cloning of Pina PPO genes Mature pineapple fruit was treated to induce brown heart disorder by maintaining the fruit for 17 days at 12 ° C and then for 4 days at 25 ° C. The pulp showing the symptoms of the Brown heart was dissected from the fruit, frozen in liquid nitrogen and ground into a fine powder in a pre-cooled coffee grinder. To isolate total RNA, 10 g were ground of the powder in a mortar and pestle, then extracted with 30 ml of homogenization buffer (100 mM Tris-HCl, pH 9.0, 200 mM NaCl, 15 mM EDTA, 0.5% sarkosyl and 1% β-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 subjected to centrifugation for 15 minutes at 8,000 RPM and 4 ° C. The aqueous phase top was removed, and an equal volume of isopropanol was added. The mixture was incubated for 30 minutes at -70 ° C, then subjected to centrifugation for 20 minutes at 8,000 RPM and 4 ° C in Corex tubes. The supernatant was removed, and the pellet was rinsed with 70% ethanol and subjected to centrifugation for 5 minutes at 8,000 RPM and 4 ° C. The ethanol was removed, and the pellet was air dried, then resuspended in 0.75 ml of sterile water, and subjected to centrifugation to remove any insoluble material. LiCl was added to a final concentration of 3 M, and the mixture was incubated overnight at -20 ° C, then subjected to centrifugation for 30 minutes at 15,000 RPM and 4 ° C. The pellet was rinsed with 70% ethanol , it was briefly subjected to centrifugation, drained and dried at air. The pellet was resuspended in 75 μl of sterile water, and subjected to centrifugation to remove any insoluble material. Ig-nucleotide primers were designed based on known plant PPO DNA sequences. Comparison of a number of PPO sequences from a range of different plants allowed the identification of the conserved regions of the gene, which are mainly in or near the regions that encode the two copper binding sites, CuA and CuB. The forward primers designed around the CuA site (GEN8, GEN9 and GENIO) and the reverse primers designed around the CuB site (REVI and REV2) were synthesized: GEN8: (5'-GCGAATTCGATCCIACITT [TC] GC [GT] TTICC-3 ') GEN9: (5'-GCGAATTCTICA [TC] TG [TC] GCIT [TC] TG-3') GENE: (5'-GCGAATTCTTICCIT [TA] [TC] TGGA [TC] - TGGG-3 ') REVI: (5 '-GCCTGCAGCCACATIC [TG] [AG] TCIAC [AG] TT-3') REV2: (5'-GCCTGCAGTT [TC] TC [AG] TC [AG] TAGAA-3 ') The first-strand cDNA was synthesized from 10 μg of total RNA with reverse transcriptase as described in reference 2, using the REV2 primer: REV2: (5'-GCCTGCAGTT [TC] TC [AG] TC [AG] TAGAA-3 ') The reaction of the first strand was amplified by the polymerase chain reaction (PCR) essentially according to the Frohman method (1). ), using the GEN and REV primers described in Example 1, each in a final concentration of 1 μM (2). Amplification involved an initial program of 2 cycles of denaturation at 94 ° C for 1 minute, annealing at 37 ° C for 2 minutes, a slow slope at 72 ° C for 2 minutes and elongation at 72 ° C for 3 minutes, followed by 33 cycles of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and elongation at 72 ° C for 3 minutes. 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 agarose gel, and the bands of interest were cut. The DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen). The purified DNA was cloned into the Bluescript SK * vector cut with Eco-RV (Stratagene) to which T was placed at the tail end with Taq Polymerase, and the ligated DNA was introduced into E. coli DH5a by electroporation Recombinant clones that had an insert of the predicted size were selected and their DNA sequence was determined by automated sequence determination. A putative pineapple PPO clone (PINPPO20) was identified, based on its homology to known plant PPO genes. The first-strand cDNA was also synthesized from 10 μg of total RNA with reverse transcriptase as described in Dry, IB and Robinson, SP (1994), using an oligo-dT initiator adapter (Reference 1): B26: ( 5 '-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3'). This first-strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman, MA (1990) using GEN9 primers and GENIO: GEN9: (5 '-GCGAATTCTICA [TC] TG [TC] GCITA [TC] TG-3 ') GENE: (5'-GCGAATTCTTICCIT [TA] [TC] TGGAA [TC] -TGGG-3') in a final concentration of 1 μM and an adapter primer B25: _ B25: (5 '-GACTCGAGTCGACATCGA-3') in a final concentration of 0.1 μM (Frohman, MA (1990), Dry, IB and Robinson, SP (1994)). The amplification involved a program of 33 cycles of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and elongation at 72 ° C for 3 minutes. 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 agarose gel, and the bands of interest were cut. The DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen). The purified DNA was cloned into the Bluescript SK * vector cut with Eco-RV (Stratagene) to which T was placed at the tail end with Taq Polymerase, and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones that had an insert of the predicted size were selected and their DNA sequence was determined by automated sequence determination. Two putative pineapple PPO clones (PINPPOl and PINPP02) were identified, based on their homology to known plant PPO genes. It was found that the sequence of PINPPOl was almost identical to that of PINPPO20. The 5 'end of PINPPOl was obtained using a 5' -RACE system for rapid amplification of End of cDNA, Version 2.0, of GIBCO-BRL, according to the manufacturer's instructions. Specific oligonucleotide primers were used, based on the sequences of PINPPO1 and PINPP02: PINE1: (SEQ ID NO: 29): 5'-ATATCACCTGTCGGTACATGACGGC - PINE2: (SEQ ID NO: 30): 5 '- GTGCCATTGTAGTCGAGGTCAATCAr 3' The sequence was determined to a number of clones and it was found that one, 5PINA, was almost identical to PINPPOl in the regions of overlap. A natural-length pineapple cDNA clone was isolated, using a primer designed to the 5 'end sequence of 5PINA: "" 5PXNT: (SEQ ID NO: 31): (5'-CCAGTGCCTGGTTTAGGTGTATTCAC-3') were used with the B25 adapter initiator as described above to amplify cDNA prepared from RNA of pineapple fruit to which the brown heart was induced. The amplification involved a program of 33 cycles of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and elongation at 72 ° C for 3 minutes. A sample of the amplified DNA was run on an agarose gel, and stained with ethidium bromide to determine the size of the products of the PCR. The remainder was run on a low melting agarose gel, and the bands of interest were cut. The DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen). 'The purified DNA was cloned into the vector Bluescript SK * cut with Eco-RV (Stratagene) to which T was placed at the tail end with Taq Polymerase, and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones that had an insert of the size "predicted (2.2 kbp) were selected and their DNA sequence was determined by automated sequence determination.A clone of pineapple PPO (PINPPOFL) was identified, based on its homology to the clones. PINPPO20, PINPPOl and 5PIN It was found that the PINPPOFL sequence was almost identical to that of PINPPO20, PINPPOl and 5PINA in the overpopulation regions GENERAL Those skilled in the art will be aware that the invention described herein is subject to variations and modifications different from those specifically described, it is to be understood that the invention described herein includes all variations and modifications. The invention also includes all the steps, aspects, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or aspects. Throughout this specification, unless the context otherwise requires the word "comprises", and variations such as "comprises" and "comprising", it will be understood that it implies the inclusion of an integer or set stage or group of integers or stages, but not the exclusion of any other integer or stage or group of integers or stages. REFERENCES 1. Frohman, MA (1990) in PCR Protocols: A Guide to Methods and Applications "(PCR Protocols: A Guide to Methods and Applications) (MA Innis, DH Gelfrand, JJ Sninsky and TJ White, editors) Academic Press, New York pages 28-38 2. Dry, IB and Robinson, SP (1994) "Molecular Cloning and Characterization of grape berry polyphenol oxidase" (Molecular Cloning and Characterization of grape fruit polyphenol oxidase) Plant Mol. Biol. , 495-502.

LIST OF SEQUENCES < 11D > SCIENTIFIC AND INDUSTRIAL COMMUNITY RESEARCH ORGANIZATION < 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 < 1S0 > 33 < 170 > Patent in Ver. 2 . 0 < 211 > 582 < 212 > DNA < 213 > banana < 220 > < 221 > CDS < 11? 1 > (1) .. (582) < 400 > 1 cac tgt gcg tat tgt gat ggc gcc tac gac cag atc ggc ttc ccc aac 48 His Cys Wing Tyr Cys Asp Gly Wing Tyr Asp Gln lie Gly Phe Pro Asn 1 5 10 15 ctc gag ctc cac gtc cac aac tcc tgg ctc ttc tc cct tgg cac cgc 96 Leu Glu Leu Gln Val His Asn Ser Trp Leu Phe Phe Pro Trp His Arg 20 25 30 ttc tac ctc tac tcc cac gag agg atc ctc gga aag ctc ata ggc gac 144 Phe Tyr Leu Tyr Phe His Glu Arg lie Leu Gly Lys Leu lie Gly Asp 35 40 45 gac act tcc gcc ctc cct ttc tgg aac tgg gac gcg ccc ggc ggc atg 192 Asp Thr Phe Wing Leu Pro Phe Trp Asn Trp Asp Wing Pro Gly Gly Met 50 55 60 aag ctg ccg tcg atc tac gcc gac cct tcg tcc tcg ctc tat gac aag 240 Lys Leu Pro Ser lie Tyr Wing Aep Pro Ser Ser Leu Tyr Asp Lys 65 70 75 80 ttt cgc gac gcc aag cac cag ccg cea gtc ctc gtc gac ctc gac tac 288 Phe Arg Asp Ala Lys His Gln Pro Pro Val Leu Val Asp Leu Asp Tyr 85 90 95 aac gga acc gac cct agt ttc acc gac gca gag cag atc gat cag aac 336 Asn Gly Thr Asp Pro Ser Phe Thr Asp Ala Glu Gln lie Asp Gln Asn 100 105 110 ctc aag atc atc tac cgg cag gtg atc tcc aac ggc aag acg ccg ttg 384 Leu Lys lie Met Tyr Arg Gln Val lie Be Asn Gly Lys Thr Pro Leu 115 120 125 ctc ttc tta ggc tcg gct tac cgt gcc ggc gac aac cea aac ccc ggc 432 Leu Phe Leu Gly Ser Wing Tyr Arg Wing Gly Asp Asn Pro Anr Pro Gly 130 135 140 gcg ggc tcg ctc gag aac ata cea cac ggc ccc gtc cag ggg tgg act 480 Wing Gly Ser Leu Glu Asn lie Pro His Gly Pro Val His Gly Trp Thr 145 150 155 - _ _Xß0 ggc gac aga age caac ccc aat ctc gag gac atg ggc aac ttc tac tcc 528 Gly Asp Arg Ser Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser 165 170 175 cg ggg c c cc cc tc atc tcc tcc gcc cac cat tea aat gcc gat ccc 576 Wing Gly Arg Asp Pro lie Phe Phe Wing His His As Asn Val Asp Arg 180 185 190 atg tgg 582 Met Trp < 210 > 2 < 211 > 194 < 212 > PRT < 212 > banana < 400 > 2 His Cys Wing Tyr Cys Asp _Gly Wing Tyr Asp Gln He Gly Phe Pr_ Asn 1 5 10 15-- Leu Glu Leu Gln Val His Asn Ser Trp Leu Phe Phe Pro Trp His Arg 20 25 30 Phe Tyr Leu Tyr Phe His Glu Arg He Leu Gly Lys Leu He Gly Asp 35 40 45 Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Wing Pro Gly Gly Met 50 55 60 Lys Leu Pro Ser He Tyr Wing Asp Pro Ser Ser Leu Tyr Asp Lys 65 70 75 80 Phe Arg Asp Wing Lys His Gln Pro Pro Val Leu Val Asp Leu Asp Tyr 85 90 95 Asn Gly Thr Asp Pro Be Phe Thr Asp Wing Glu Gln He Asp Gln Asn 100 105 110 Leu Lys He Met Tyr Arg Gln Val He Ser Asn Gly Lys Thr Pro Leu 115 120 125 Leu Phe Leu Gly Ser Wing Tyr Arg Wing Gly Asp Asn Pro Asn Pro Gly 130 135 140 Wing Gly Ser Leu Glu Asn lie- Pro His Gly Pro Val His Gly Trp Thr 145 150 155 __160 Gly Asp Arg Ser Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser 165 170 175 Wing Gly Arg Asp Pro He Phe Phe Wing His His As Asn Val Asp Arg 180 185 190 Met Trp < 210 > 3 < 21I > 426 < 212 > DNA < 213 > banana < 220 > < 221 > CDS < 222 > (1) .. (426) < 400 > 3 ttg ccg ttt tgg aat tgg gac gcg ccc ggc ggc atg aag ctg ccg tcg 48 Leu Pro Phe Trp Asn Trp Asp Wing Pro Gly Gly Met Lys Leu Pro Ser 1 5 10 15 atc tac gcc gac cct tcg tcc tcg ctc tat gac aag ttt cgc gac gcc 96 He Tyr Wing Asp Pro Ser Ser Leu Tyr Asp Lys Phe Arg Asp Wing 20 25 30 aag cac cg gc ccg ccc gtc gac ctc gac tac aac gga acc gac 144 Lys His Gln Pro Pro Val Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp 35 40 45 cct agt ttc acc gac gca gag cag atc gat cag aac ctc aag atc atg 192 Pro Ser Phe Thr Asp Ala Glu Gln He Asp Gln Asn Leu Lys He Met 50 55 60 tac cgg cag gtg atc tcc aac ggc aag acg ccg ttg ctc ttc tta ggc 240 Tyr Arg Gln Val He Ser Asn Gly Lys Thr Pro Leu Leu Phe Leu Gly 65 70 75 80 tcg gct tac cgt gcc ggc gac aac cea aac ccc ggc gcg ggc tcg ctc 288 Wing Wing Tyr Arg Wing Gly Asp Asn Pro Asn Pro Gly Wing Gly Ser Leu 85 90 95 gag aac ata cea cac ggc ccc gtc cac ggg tgg act ggc gac aga age 336 Glu Asn He Pro His Gly Pro Val His Gly Trp Thr Gly Asp Arg Ser 100 105 110 cac ccc aat ctc gag gac atg ggc aac ttc tac tc gcg ggg cgc gac 384 Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Arg Asp 115 1 20 125 cct atc ttc tcc gcc cac cat tea aat gtc gat age atg tgg 426 Pro He Phe Phe Wing His His As Asn Val Asp Ser Met Trp 130 135 140 < 210 > 4 < 211 > 142 < 212 > PRT < 212 > banana < 400 > 4 Leu Pro Phe Trp Asn Trp Asp Wing Pro Gly Gly Met Lys Leu Pro Ser 1 5 10 15 He Tyr Wing Asp Pro Ser Ser Leu Tyr Asp Lys Phe Arg Asp Wing 20 25 30 Lys His Gln Pro Pro Val Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp 35 40 45 Pro Ser Phe Thr Asp Ala Glu Gln He Asp Gln Asn Leu Lys He Met 50 55 60 Tyr Arg Gln Val He Ser Asn Gly Lys Thr Pro Leu Leu Phe Leu Gly 65 70 75 80 Be Wing Tyr Arg Wing Gly Asp Asn Pro Asn Pro Gly Wing Gly Ser Leu 85 90 95 Glu Asn He Pro His Gly Pro Val His Gly Trp Thr Gly Asp Arg Ser 100 105 110 Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser Wing Gly Arg Asp 115 120 125 Pro He Phe Phe Wing His His As Asn Val Asp Ser Met Trp 130 135 140 < 210 > 5 < 211 > 925 < 212 > DNA < 213 > banana <; 220 > < 221 > CDS < 222 > (2) .. (853) < 400 > 5 g ttg ctc ttc tta ggc tcg gct tac cgt gcc gcc gac aac cea aac ccc 49 Leu Leu Phe Leu Gly Ser Wing Tyr Arg Wing Gly Asp Asn Pro Asn Pro 1 5 10 15 ggc gcg ggc tcg ctc gag aac ata cea cac ggc ccc gtc cac ggg tgg 97 Gly Wing Gly Ser Leu Glu Asn He Pro His Gly Pro Val His Gly Trp 20 25 30 act ggc gac aga aac c a c c c a ct c gac gac at g gc aac tcc tac 145 Thx Gly Asp Arg Asn Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr 35 40 45 tcc gcg ggg cgc gac cct atc ttc tcc gcc cac cat tea aac gtc gac 193 Ser Wing Gly Arg Asp Pro He Phe Phe Wing His Ser Asn Val Asp 50 55 60 cgc atg tgg tac tgg tgg aag agg 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 65 70 75 80 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 85 90 95 gct gac tta gtt cga gtc acc ctc aag gac tgc ttg cag ccg gag tgg 337 Wing Asp Leu Val Arg Val Thr Leu Lys Asp Cys Leu Gln Pro Glu Trp 100 105 110 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 He Pro Trp Leu Lys Thr Arg 115 120 125 ccg act ccc aaa gcc ttg aag gcg cag aaa acc gca gcg aaa here ctg 433 Pro Thr Pro Lys Wing Leu Lys Wing Gln Lys Thr Wing Wing Lys Thr Leu 130 135 140 aaa gct here gca gag acg ccg ttc ceg gtg acg ctg cac tcc gcg gtg 481 Lys Wing Thr Wing Glu Thr Pro Phe Pro Val Thr Leu Gln Ser Wing Val 145 150 155 160 age acg acg gtg agg agg ccc aag gta tcg agg age ggc aag gag_ aag 529 Ser "Thr Thr Val Arg Arg Pro Lys Val Ser Arg Ser Gly Lys Glu Lys 165 170 175_. gaa gag gag gag gac gtc ctc atc gtg gag ggg gac gac tc gac cgc 577 Glu Glu Glu Glu Glu Val Leu He Val Glu Gly He Glu Phe. Asp Arg 180 185 190 gac tac ttc ata aag ttc gac gtc ttc gtg aac gcc acc. gag ggt gag 625 Asp Tyr Phe He Lys Phe Asp Val Phe Val Asn Wing Thr Glu Gly Glu 195 200 205 ggc atc acg ccg ggc gcc age gag ttc gcg ggc age ttc gtc aac gtc 673 Gly He Thr Pro Gly Ala Ser Glu Phe Ala Gly Ser Phe Val Asn Val 210 215 220 ccg cac aag cac aag cac age aag aag aag aag aag acag agg 721 Pro His Lys His Lys His Ser Lys Lys Glu Lys Lys Leu Lys Thr Arg 225 230 235 - 240 ctc tgc ctg ggg atc act gac ctg ctc gag gac atc ggg gcg gag gac 769 Leu Cys Leu Gly He Thr Asp Leu Leu Glu Asp He Gly Wing Glu Asp 245 250 255 gac gac age gtc gtc ctc gtc acc atc ccg aaa gcc gga aag ggc aag 817 Asp Asp Ser Val Leu Val Thr He Val Pro Lys Wing Gly Lys Gly Lys 260 265 270 gtg tcg gtc ctc ggc cgc atc gcc gat aat ttc cea tgaagtaata Val Ser 863 Val Ala Asp Gly Leu Arg Phe Pro Asn He 275 280 ctactaccta ctatatattt tcaaggaaaa taaaagccgc accatcgtaa caaaaaaaaa 923 aa 925 < 210 > 6 < 211 > 284 < 212 > PRT < 211 > banana < 400 > 6 Leu Leu Phe Leu Gly Be Wing Tyr Arg Wing Gly Asp Asn Pro Asn "Pro 1 5 10 15 Gly Wing Gly Ser Leu Glu Asn He Pro His Gly Pro Val His GIV Trp 20 25 30 Thr Gly Asp Arg Asn Gln Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr 35 40 45 Be Wing Gly Arg Asp Pro He Phe Phe Wing His His As Asn Val Asp 50 55 60 Arg Met Trp Tyr Leu Trp Lys Lys Leu Gly Gly Lys His Gln Asp Phe 65 70 75 80 Asn Asp Lys Asp Trp Leu Asn Thr Thr Phe Leu Phe Tyr Asp Glu Asn 85 90 95 Wing Asp Leu Val Arg Val Thr Leu Lys Asp Cys Leu Gln Pro Glu Trp 100 105 110 Leu Arg Tyr Asp Tyr Gln Asp Val Glu He Pro Trp Leu Lys Thr Arg 115 120 125 Pro Thr Pro Lys Wing Leu Lys Wing Gln Lyt Thr Wing Wing Lys Thr Leu 130 135 140 Lys Ala Thr Ala Glu Thr Pro Phe Pro Val Thr Leu Gln Ser Ala Val 145 150 155 -160 Be Thr Thr Val Arg Arg Pro Lys Val Ser Arg Ser Gly Lys Glu Lys 165 170 175 Glu Glu Glu Glu Glu Val Leu He Val Glu Gly He Glu Phe Asp Arg 180 185 190 Asp Tyr Phe He Lys Phe Asp Val Phe Val Asn Ala Thr Glu Gly Glu 195 200 205 Gly He Thr Pro Gly Wing Ser Glu Phe Wing Gly Ser Phe Val Asn Val 210 215 220 Pro His Lys His Lys His Ser Lys Lys Glu Lys Lys Leu Lys Thr Arg 225 230 235 240 Leu Cys Leu Gly He Thr Asp Leu Leu Glu Asp He Gly Wing Glu Asp 245 250 255 Asp Asp Ser Val Leu Val Thr He Val Pro Lys Wing Gly Lys Gly Lys 260 265 270 Val Ser Val Wing Gly Leu Arg He Asp Phe Pro Asn ~~ 275 280 < 210 > 7 < 211 > 960 < 212 > DNA < 213 > banana < 220 > < 221 > CDS < 222 > (2) .. (853) < 4O0 > 7 g ttg ctc ttc tta ggc tcg gct 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 1 May 10 15 ggc gcg gga aka atc gag aac atg ccg cac aac aac gtg cac ttg tgg 97 Gly Wing Gly Ser He Glu Asn Met Pro His Asn Asn Val His Leu Trp 20 25 30 acc ggc gac cgc acc ccacc ccc aac tcc gag aac atg ggc acc ttc tac 145 Thr Gly Asp Arg Thr Gln Pro Asn Phe Glu Asn Met Gly Thr Phe Tyr 35 40 45 gcg gcg gcg gac ccc atc ttc tcc gcc cac gcc aac atc gac 193 Ala Ala Ala Arg Asp Pro He Phe Phe Wing His His Wing Asn He Asp 50 55 60 cga atg tgg tac ctg tgg aag aag ctc age agg aag cac cag gac ttc 241 Arg Met Trp Tyr Leu Trp Lys Lys Leu Ser Arg Lys His Gln Asp Phe 65 70 75 80 aat gac tcg gac tgg ctc aaa gct 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 85 90 95 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 100 105 110 ctc cgc tac acg tac cac gac gtg aag atc ce tgg gcg aac acccga 385 Leu Arg Tyr Thr Tyr Gln Asp Val Lys He Pro Trp Wing Asn Thr Arg 115 120 125 ccg act ccc aag ctc gcc aag gcg agg aaa gcc ggc age aga tcg ctg 433 Pro Thr Pro Lys Leu Wing Lys Wing Arg Lys Wing Gly Ser Arg Ser Leu 130 135 140 _ 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 145 150 155 _160 aaa gtg acg gtg aag agg ccc aag gtg ggg agg age ggc aag gag ^ aag 529 Lys Val Thr Val Lys Arg Pro Lys Val Gly Arg Ser Gly Lys Glu Lys 165 170 17S_ gaa gat gag gag gait ata ctc ata gtg gag ggg atc gag tcc gac cgc 577 Glu Asp Glu Glu Glu He Leu He Val Glu Gly He Glu Phe Asp Arg 180 185 190 gac tac tc atc aag tcc gac gtc tte gtg aac gcg acg gag ggc_ gac 625 Asp Tyr Phe He Lys Phe Asp Val Phe Val Asn Ala Thr Glu Gly Asp 195 200 205 ggc atc acg gcc ggg gcc agt gag ttc gcc ggc age ttc gtg aac gtc 673 Gly He Thr Ala Gly Wing Ser Glu Phe Wing Gly Ser Phe Val Asn Val 210 215 220 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 225 230 235 240 ctg tgt ctg gga atc acc gac ctg ctc gag gac atc ggc gcg gag gac 769 Leu Cys Leu Gly He Thr Asp Leu Leu Glu Asp He Gly Ala Glu Asp 245 250 255 gac gac age gtg ctc gtc acc atc gtg ccg aag gca ggc aaa gga aag 817 Asp Asp Ser Val Leu Val Thr He Val Pro Lys Ala Gly Lys Gly Lys 260 265 270 gtg aka gtc ggc ggt ctt cgg att ttt gac aag aka tgaggaaata Val Ser Val 863 Gly Gly Leu Ser Phe Asp Arg He Lys 275 280 aaagaattea cgtgccgtgc ctgctttcaa tgtacgaatg aaataagagt gcatcatcac aaaaaaaaaa aaaaaaa 923 tctactttaa cgaccatggt 960 < 210 > 8 < 211 > 284 < 212 > PRT < 213 > banana < 400 > 8 Leu Leu Phe Leu Gly Be Wing Tyr Arg Wing Gly Asp Gln Pro Asn Pro 1 5 10 15 Gly Wing Gly Ser He Glu Asn Met Pro His Asn Asn Val His Leu Trp 20 25 30 Thr Gly Asp Arg Thr Gln Pro Asn Phe Glu Asn Met Gly Thr Phe Tyr 35 40 45 Wing Wing Arg Asp Pro He Phe Phe Wing His His Wing Asn He Asp 50 55 60 Arg Met Trp Tyr Leu Trp Lys Lys Leu Ser Arg Lys His Gln Asp Phe 65 70 75 80 Asn Asp Ser Asp Trp Leu Lys Wing Being Phe Leu Phe Tyr Asp Glu Asn 85 90 95 Wing Asp Leu Val Arg Val Thr Val Lys Asp Cys Leu Glu Thr Glu Trp 100 105 110 Leu Arg Tyr Thr Tyr Gln Asp Val Lys He Pro Trp Wing Asn Thr Arg 115 120 125 Pro Thr Pro Lys Leu Wing Lys Wing Arg Lys Wing Gly Ser Arg Ser Leu 130 135 140 Tys Ala Thr Ala Glu Val Gln Phe Pro Val Thr Leu Glu Ser Pro Val 145 150 155 - -160 Lys Val Thr Val Lys Arg Pro Lys Val Gly Arg Ser Gly Lys Glu Lys 165 170 175 Glu Asp Glu Glu Glu He Leu He Val Glu Gly He-Glu Phe Asp Arg 180 185 190 Asp Tyr Phe He Lys Phe Asp Val Phe Val Asn Wing Thr Glu Gly Asp 195 200 205 Gly He Thr Wing Gly Wing Ser Glu Phe Wing Gly Ser Phe Val Asn Val 210 215 220 Pro His Lys His Lys His Arg Lys Asp Glu Asn Lys Leu Lys Thr Arg 225 230 235 240 Leu Cys Leu Gly He Thr Asp Leu Leu Glu Asp He Gly Wing Glu Asp 245 250 255 Asp Asp Ser Val Leu Val Thr He Val Pro Lys Wing Gly Lys Gly Lys 260 265 270 Val Ser Val Gly Gly Leu Arg He Asp Phe Ser Lys 275 280 < 210 > 9 < 211 > 545 < 212 > DNA < 213 > tobacco < 220 > < 221 > CDS < 222 > (1) .. (543) < 400 > 9 gat ccg acg ttt gcg ttg cea tat tgg aac tgg gat cat cea aag ggc 48 Asp Pro Thr Phe Wing Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly 1 5 10 15 atg cgt ttg cea cac atg ttt gat caa cea aac gtg tac cct gat ctt 96 Met Arg Leu Pro His Met Phe Asp Gln Pro Asn Val Tyr Pro Asp Leu 20 25 30 tac gat cea aga cgt aac caa gaa cac cgc ggt tet gta atc atg gac 144 Tyr Asp Pro Arg Arg Asn Gln Glu His Arg Gly Ser Val He Met Asp 35 40 45 ctt ggt cat ttt ggt ca gc ga gtg aaa gga act gac ttg ca atg atg 192 Leu Gly His Phe Gly Gln Asp Val Lys Gly Thr Asp Leu Gln Met Met 50 55 60 age aat aac ctt act cta atg tat cgt caa atg att acc aat tea cea 240 Ser Asn Asn Leu Thr Leu Met Tyr Arg Gln Met He Thr Asn Ser Pro 65 70 75 80 tgt cea caa ctc ttt ttc ggt aag cea tat tgt acg gaa gtt gga ccc 288 Cys Pro Gln Leu Phe Phe Gly Lys Pro Tyr Cys Thr Glu Val Gly Pro 85 90 95 aaa cea ggg cag gga gct att gaa aac atc cct cat act cct gtc cac 336 Lys Pro Gly Gln Gly Wing He Glu Asn He Pro His Thr Pro Val His 100 105 110 att tgg gtt ggt agt aag cct aat gag aat aac tgt aaa aac ggt gaa 384 He Trp Val Gly Ser Lys Pro Asn Glu Asn Asn Cys Lys Asn Gly Glu 115 120 12-5 gat atg gga aat ttc tat gct gctt gctt aag gat cct gct ttc tat agt 432 Asp Met Gly Asn Phe Tyr Ser Wing Gly Lys Asp Pro Wing Phe Tyr Ser 130 135 140 cac cat gca aat gta gat cgc atg tgg here ata tgg aaa here tga gga 480 His His Wing Asn Val Asp Arg Met Trp Thr He Trp Lys Thr Leu Gly 145 150 155 _160 gga aaa cgc aag gac atc aac aag cea gat tat ttg aac act gag_ ttc 528 Gly Lys Arg Lys Asp He Asn Lys Pro Asp Tyr Leu Asn Thr Glu Phe 165 170 175 ttt ttc tac gac gaa aa 545 Phe Phe Tyr Asp Glu 180 < 210 > 10 < 211 > 181 < 212 > PRT < 213 > tobacco < 400 > 10 Asp Pro Thr Phe Wing Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly 1 5 10 15 Met Arg Leu Pro His Met Phe Asp Gln Pro Asn Val Tyr Pro Asp Leu 20 25 30 Tyr Asp Pro Arg Arg Asn Gln Glu His Arg Gly Ser Val He Met Asp 35 40 45 Leu Gly His Phe Gly Gln Asp Val Lys Gly Thr Asp Leu Gln Met Met 50 55 60 Ser Asn Asn Leu Thr Leu Met Tyr Arg Gln Met He Thr Asn Ser Pro 65 70 75 80 Cys Pro Gln Leu Phe Phe Gly Lys Pro Tyr Cys Thr Glu Val Gly Pro 85 90 95 Lys Pro Gly Gln Gly Wing He Glu Asn He Pro His Thr Pro Val His 100 105 110 He Trp Val Gly Ser Lys Pro Asn Glu Asn Asn Cys Lys Asn Gly Glu 115 120 125 Asp Met Gly Asn Phe Tyr Ser Wing Gly Lys Asp Pro Wing Phe Tyr Ser 130 135 140 His His Wing Asn Val Asp Arg Met Trp Thr He Trp Tys Thr Leu Gly 145 150 155 -160 Gly Lys Arg Lys Asp He Asn Lys Pro Asp Tyr Leu Asn Thr Glu Phe 165 __ 170 175 Phe Phe Tyr Asp Glu 180 < 210 > 11 < 211 > 673 < 212 > DNA < 213 > tobacco < 220 > < 221 > CD? < 222 > (3) .. (671) < 400 > 11 tg cac tgt gcr tat tgc aac ggt gct tac aaa att ggt ggc aaa gag 47 His Cys Wing Tyr Cys Asn Gly Wing Tyr Lys He Gly Gly Lys Glu 1 5 10 -15 tta cea 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 20 25 30 ttg tac ttc tat gaa aga atc ttg ggc tet tta att aat gat cct act 143 Leu Tyr Phe Tyr Glu Arg He Leu Gly Ser Leu He Ash Asp PrcTThr 35 40 45 ttt ggt ttg cea tat tgg aac tgg gac cat cea aag ggc atg cgt ata 191 Phe Gly Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly Met Arg He 50 55 60 cct cec atg ttc gat cgt gaa ggg tet tcc ctt tac gac gaa aaa cgt 239 Pro Pro Met Phe Asp Arg Glu Gly Ser Ser Leu Tyr Asp Glu Lys Arg 65 70 75 aac cata agt cac cgt aat gga acc ata att gat ctt ggt cat ttc ggt 287 Asn Gln Ser His Arg Asn Gly Thr He He Asp Leu Gly His Phe Gly 80 85 90 95 caa gaa gtc caca here act ca g t g cag at g actg act aat aac tta act 335 Gln Glu Val Gln Thr Thr Gln Leu Gln Gln Met Thr Asn Asn Leu Thr 100 105 110-ata atg tat cgt ca at at ata ata aat gct cct tgc ccc ttg ctc ttc 383 He Met Tyr Arg Gln Met He Thr Asn Wing Pro Cys Pro Leu Leu Phe 115 120 125 ttt ggt cag cct tac cct cta cta gga act gat cctc agt cea ggg atg ggc 431 Phe Gly Gln Pro Tyr Pro Leu Gly Thr Asp Pro Pro Gly Met Gly 130 135 140 act att gaa aac atc cct cat act cct gtc cac att tgg gtt gt agt 479 Thr He Glu Asn He Pro His Thr Pro Val His He Trp Val Gly Ser 145 150 155 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 160 165 170 175 tac tcg gcc gta tta gac ccg ctt ttc tat tcc cat cac gcc aat gtg 575 Tyr Ser Wing Gly Leu Asp Pro Leu Phe Tyr Ser His His Wing Asn Val 180 185 190 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 195 200 205 ctc acg cac aaa gat tgg ttg aac ttc gag ttc ttt ttc tac gat gaa 671 Leu Thr His Lys Asp Trp Leu Asn Ser Glu Phe Phe Phe Tyr Asp Glu 210 215 220 673 < 210 > 12 < 211 > 223 < 212 > PRT < 213 > tobacco < 400 > 12 His Cys Ala Tyr Cys Asn Gly Ala Tyr Lys He Gly Gly Lys Glu Leu 1 5 10 15- Gln Val His Phe Ser Trp Leu Phe Phe Pro Phe His Arg Trp Tyr Leu 20 25 3-0 Tyr Phe Tyr Glu Arg He Leu Gly Ser Leu He Asn Asp Pro Thr Phe 35 40 45 Gly Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly Met Arg He Pro 50 55 60 Pro Met Phe Asp Arg Glu Gly Ser Ser Leu Tyr Asp Glu Lys Arg Asn 65 70 75 80 Gln Ser His Arg Asn Gly Thr He He Asp Leu Gly His Phe Gly Gln 85 90 95 Glu Val Gln Thr Thr Gln Leu Gln Gln Met Thr Asn Asn Leu Thr He 100 105 110 Met Tyr Arg Gln Met He Thr Asn Ala Pro Cys Pro Leu Leu Phe Phe 115 120 125 Gly Gln Pro Tyr Pro Leu Gly Thr Asp Pro Ser Pro Gly Met Gly Thr 130 135 140 He Glu Asn He Pro His Thr Pro Val His He Trp Val Gly Ser Arg 145 150 155 160 Leu Asp Glu Asn Asn Thr Lys Hie Gly Glu Asp Met Gly Asn Phe Tyr 165 170 175 Be Wing Gly Leu Asp Pro Leu Phe Tyr Ser His His Wing Asn Val Asp 180 185 190 Arg Met Trp Ser Glu Trp Lys Ala Leu Gly Gly Lys Arg Arg Asp Leu 195 200 205 Thr His Lys Asp Trp Leu Asn Ser Glu Phe Phe Phe Tyr Asp Glu 210 215 220 < 210 > 13 < 21X > 685 < 212 > DNA < 213 > tobacco < 220 > < 221 > CDS < 222 > (3) .. (683) < 4Q0 > 13 tg cat tgt gcg tat tgc aac gat gct tac here atg ggt gac caa aag 47 His Cys Wing Tyr Cys Asn Asp Wing Tyr Thr Met Gly Asp Gln Lys 1 5 10 -15 tta ca gtt cac tcg tgg ctt ttc ttc ccg ttt cat aga tgg tac 95 Leu Gln Val His Gln Ser Trp Leu Phe Phe Pro Phe His Arg Trp Tyr 20 25 30 ttg tac tcc tac gag aga atc ttg gcc tcc ctc atc gat gat cea act 143 Leu Tyr Phe Tyr Glu Arg He Leu Gly Ser Leu He Asp Asp Pro Thr 35 40 45 ttt gct ctg cea tat tgg aac tgg gac cat cea age ggc atg cgt ttg 191 Phe Wing Leu Pro Tyr Trp Asn Trp Asp His Pro Ser Gly Met Arg Len 50 55 60 cct gct atg ttc gat gtc gaa ggt tet tcc ctc tac gat gca aga cgt 239 Pro Wing Met Phe Asp Val Glu Gly Ser Ser Leu Tyr Asp Wing Arg Arg 65 70 75 aat cea cat gtc cgt aat gga acc atc gat ctt ggt ttt ttc ggt 287 Asn Pro His Val Arg Asn Gly Thr He He Asp Leu Gly Phe Phe Gly 80 85 90 95 gat gaa gtc aaa act aat gaa ata cag atg ata act aac aac aac tta att 335 Asp Glu Val Lys Thr Asn Glu He Gln Met He Thr Asn Asn Leu He 100 105 110 cta atg tat cgt ca g at t ata g t tc tc ccg ctg ttg ttc 383 Leu Met Tyr Arg Gln Met He Thr Asn Wing Pro Cys Pro Leu Leu Phe 115 120 125 ttc gga gag cct tac aga tga tga aga cta aat ccg ggg cag gga 431 __ Phe Gly Glu Pro Tyr Arg Phe Gly Ser Lys Pro Asn Pro Gly Gln Gly 130 135 140 acc att gaa aac att cct cat act ccg gtt cac att tgg act ggt act 479 Thr He Glu Asn He Pro His Thr Pro Val His He-Trp Thr Gly Thr 145 150 155 gtg cgg tgt acg gat ttg ggt aat tgt gtg cea tea tac ggt gag gat 527 Val Arg Cys Thr Asp Leu Gly Asn Cys Val Pro Ser Tyr Gly Glu Asp 160 165 170 175 atg ggt aat ttc tac tea gct ggt tta gac cea gtt ttt tac age cac 575 Met Gly Asn Phe Tyr Be Ala Gly Leu Asp Pro Val Phe Tyr Ser His 180 185 190 cac gcc aat gtg gac cgc atg tgg aat gaa tgg aaa gca cta gga ggg 623 His Ala Asn Val Asp Arg Met Trp Asn Glu Trp Lys Ala Leu Gly Gly 195 200 205 aaa aga agg gat ctc here gac aat gat tgg tta aac tcg gag ttc ttt 671 Lys Arg Arg Asp Leu Thr Asp Asn Asp Trp Leu Asn Ser Glu Phe Phe 210 215 220 ttc tac gac gaa aa 685 Phe Tyr Asp Glu 225 < 2LD > 14 < 211 > 227 < 212 > PRT < 213 > tobacco < 400 > 14 His Cys Wing Tyr Cys Asn Asp Wing Tyr Thr Met Gly Asp Gln Lys Leu 1 5 10 15 Gln Val His Gln Ser Trp Leu Phe Phe Pro Phe His Arg Trp Tyr Leu 20 25 30 Tyr Phe Tyr Glu Arg He Leu Gly Ser Leu He Asp Asp Pro Thr Phe 35 40 45 Wing Leu Pro Tyr Trp Asn Trp Asp His Pro Ser Gly Met Arg Leu Pro 50 55 60 Wing Met Phe Asp Val Glu Gly Ser Ser Leu Tyr Asp Ala Arg Arg Asn 65 70 75 80 Pro His Val Arg Asn Gly Thr He He Asp Leu Gly Phe Phe Gly Asp 85 90 95 Glu Val Lys Thr Asn Glu He Gln Met He Thr Asn Asn Leu He Leu 100 105 110 Met Tyr Arg Gln Met He Thr Asn Wing Pro Cys Pro Leu Leu Phe Phe 115 120 125 Gly Glu Pro Tyr Arg Phe Gly Ser Lys Pro Asn Pro Gly Gln Gly Thr 130 135 140 He Glu Asn He Pro His Thr Pro Val His He Trp Thr Gly Thr Val 145 150 155 -. -160 Arg Cys Thr Asp Leu Gly Asn Cys Val Pro Ser Tyr Gly Glu Asp Met 165 170 175 Gly Asn Phe Tyr Ser Wing Gly Leu Asp Pro Val Phe Tyr Ser His His 180 185 190 Wing Asn Val Asp Arg Met Trp Asn Glu Trp Lys Wing Leu Gly Gly Lys 195 200 205 Arg Arg Asp Leu Thr Asp Asn Asp Trp Leu Asn Ser Glu Phe Phe Phe 210 215 220 Tyr Asp Glu 225 < 210 > 15 < 211 > 670 < 212 > DNA < 213 > pineapple < 220 > < 221 > CDS < 222 > (3) (668) < 400 > fifteen tg cat tgt gcg tac tgc gac ggc gcg tat gac cac atc ggc tcc ccc 47 His Cys Wing Tyr Cys Asp Gly Wing Tyr Asp Gln He Gly Phe Pro 1 5 10 __as gat ctc gag atc cag atc cac aac tcg tgg ctc ttc ttt cct tgg cac 95 Asp Leu Glu He Gln He His Asn Ser Trp Leu Phe Pro Trp His 20 25 30 cgg ttc tac ctc tac tc aac gag cgc ata ctc ggg aaa ctt atc ggc 143 Arg Phe Tyr Leu Tyr Phe Asn Glu Arg He Leu Gly Lys Leu He Gly 35 40 45 gac gac acg tcc gcg ctg cct tcc tgg aac tgg gac gcg ccg ggg gcc 191 Asp Asp Thr Phe Wing Leu Pro Phe Trp Asn Trp Asp Wing Pro Gly Gly 50 55 60 atg cag ttc ccg tet atc tac acg gac cct tea tcc tcg cta tat gac 239 Met Gln Phe Pro Ser He Tyr Thr Asp Pro Ser Ser Leu Tyr Asp 65 70 75 aag 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 He Asp Leu Asp 80 85 90 95 tac aat ggc acc ac ct acc ttc tcc cct gaa gaa cag att aac cac 335 Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln He Asn His 100 105 110 aac ctc gcc gtc atg tac cga cag gtg ata tcc agt gga aag here cea 383 Asn Leu Ala Val Met Tyr Arg Gln Val He Ser Ser Gly Lys Thr Pro 115 120 125 gag ctg ttt atg ggc tea 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 Asn Pro 130 135 140 ggc gca ggt tet gta gag cag aag ccg cac ggc ccg gtg cat gtg tgg 479 Gly Wing Gly Ser Val Glu Gln Lys Pro His Gly Pro Val His Val Trp 145 150 155 here 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 160 165 170 175 tcg gcg gcg tgg gac ccc gtt ttt ttc gca cac cac ggc aac atc gac 575 Ser Wing Wing Trp Asp Pro Val Phe Phe Wing His Gly Asn He Asp 180 185 190 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 195 200 205 acc gac ccc gac tgg ctc aac gcg tcc ttc ctg ttc tac gac gaa aa 670 Thr Asp Pro Asp Trp Leu Asn Wing Being Phe Leu Phe Tyr Asp Glu 210 215 220 < 210 > 16 < 211 > 222 < 212 > PRT < 213 > pineapple < 400 > 16 His Cys Wing Tyr Cys Asp Gly Wing Tyr Asp Gln He Gly Phe Pro Asp 1 5 10 15 Leu Glu He Gln He His Asn Ser Trp Leu Phe Phe Pro Trp His Arg 25 30 Phe Tyr Leu Tyr Phe Asn Glu Arg He Leu Gly Lys Leu He Gly Asp 40 45 Asp Thr Phe Wing Leu Pro Phe Trp Asn Trp Asp Wing Pro Gly Gly Met 50 55 60 Gln Phe Pro Ser He Tyr Thr Asp Pro Ser Ser Leu Tyr Asp Lys 65 70 75 80 Leu Arg Asp Ala Lys His Gln Pro Pro Thr Leu He Asp Leu Asp Tyr 85 90 95 Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln He Asn His Asn 100 105 110 Leu Ala Val Met Tyr Arg Gln Val He Ser Ser Gly Lys Thr Pro Glu 115 120 125 Leu Phe Met Gly Ser Wing Tyr Arg Wing Gly Asp Gln Pro Asp Pro_Gly 130 135 140 Wing Gly Ser Val Glu Gln Lys Pro His Gly Pro Val His Val Trp Thr 145 150 155 _ _ USE Gly Asp Arg Asn Gln Pro Asn Arg Glu Asp Met Gly Thr Leu Tyr Ser 165 170 175 Wing Wing Trp Asp Pro Val Phe Phe Wing His His Gly Asn He Asp Arg 180 185 190 Met Trp Tyr Val Trp Arg Asn Leu Gly Gly Lys His Arg Asn Phe Thr 195 200 205 Asp Pro Asp Trp Leu Asn Wing Ser Phe Leu Phe Tyr Asp Glu 210 215 220 < 210 > 17 < 211 > 1319 < 212 > DNA < 2135 pineapple < 22-0 > < 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 Wing Pro Gly Gly Met Gln He Pro Wing 1 5 10 15 atc tac gcc gac gct tcg tcc ccg ctc tac gac aag ctg cgc aat gcg 96 He Tyr Wing Asp Wing Ser Ser Pro Leu Tyr Asp Lys Leu Arg Asn Wing 20 25 30 aag cac ccg ccg act ttg gtc gac ccc gac tac aac gcc acc gac 144 Lys His Gln Pro Pro Thr Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp 35 40 45 ccg acc ttc acc cct gact cag atc gcc cac aac ctc acc atc atg 192 Pro Thr Phe Thr Pro Glu Gln Gln He Ala His Asn Leu Thr He Met 50 55 60 tac cga cag gtg ata tcc ggc ggg aag acg ccg gag ttg ttt atg ggc 240 Tyr Arg Gln Val He Ser Gly Lys Thr Pro Glu Leu Phe Met Gly 65 70 75 80 gcg gcg tac cgc gcg ggc gac gcg cea gac ccg ggc gca ggc act cta 288 Wing Wing Tyr Arg Wing Gly Asp Wing Pro Asp Pro Gly Wing Gly Thr Leu 85 90 95 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 100 105 110 ca ccc aac gac gaa gac atg ggc acg ttc tac gcg gcg gcg cgg gtac 384 Gln Pro Asn Asp Glu Asp Met Gly Thr Phe Tyr Wing Wing Wing Arg Asp 115 120 125 ccc atc ttc tcc gcc cac cc gcc aac gcc gac ccc atg tcc tac gtg 432 Pro He Phe Phe Wing His Gly Asn Val Asp Arg Met Trp Tyr Val 130 135 140 tgg cgg aaa ctc ggg ggc acg cac cgc gat tcc acc gac ccc gac tgg 480 Trp Arg Lyg Leu Gly Gly Thr His Arg Asp Phe Thr Asp Pro Asp Trp 145 150 155 160 ctc aac gcg tcc ttc ctc tcc tac gac gag aac gcg cag ctc gtc cgc 528 Leu Asn Wing Being Phe Leu Phe Tyr Asp Glu Asn Wing Gln Leu Val Arg 165 170 175 gtc aaa gta aag gac tgc ttg age gcc gac gcg ctg cgg tac acg tac 576 Val Lys Val Lys Asp Cys Leu Ser Wing Asp Wing Leu Arg Tyr Thr Tyr 180 185 190 cag gac gtc gac atc ccg tgg atc agt gcg aag ccg acg ceg aag aaa 624 Gln Asp Val Asp He Pro Trp He Be Wing Lys Pro Thr Pro Lys Lys 195 200 205 here ccg ggg ggc gct gcg cct tcc acg here gag gct ata ttt ccg gtg 672 Thr Pro Gly Gly Ala Wing Pro Ser Thr Thr Glu Wing He Phe Pro Val 2 10 215 220 gtg ctg gat aag ccg gtg age tet 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 225 230 235 240 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 He Glu Leu 245 250 255 gac aag gac gtg gcc gag aag ttc gac gtg tat ata aac gcg ccg gac 816 Asp Lys Asp Val Wing Val Lys Phe Asp Val Tyr He Asn Wing Pro Asp 260 265 270 aac gaa ggg ggg ggg cg gag gcg age gag ttc gca ggg age ttc gtc 864 Asn Glu Gly Val Gly Pro Glu Wing Ser Glu Phe Wing Gly Ser Phe Val 275 280 285 cag gtg ccg cac aag cag aag aag aag aag aag gcg agg att 912 Gln Val Pro His Lys His Lys Lys Gly Lys Lys Glu Lys Ala Arg He 290 295 300 aaa acg acg ctc agg ctc ggg ata acg gac ctg ctc gag gac atc ggc 960 Lys Thr Thr Leu Arg Leu Gly He Thr Asp Leu Leu Glu Asp He Gly 305 310 315 -320 gcc gac gac gag age gtg gtc ctc gtc acg gtg ccg agg atg ggc 100. Wing Glu Asp Asp Glu Ser Val Leu Val Thr Leu Val Pro Arg He Gly 325 330 335 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 He Asp P I Ser Lys 340 345 350 tgatcagcag caaattaact atacatgaaa gtaaaaaaaa ttgcatttac ctacctatag 1113 aagagaataa atgcgtatgt aatctgcccc atttgtcact tttaatttct cgagcgtgtt 1173 gttgcatgca ctgaatgaga tgcgcgcagc cataatgect ggtatagtgt agtagtttag 1233 gcgtggatac gtataacgta cgtatgcatg tataaggaat aaaaaaaaaa aaaaaaaaaa 1293 aatgatgagt ttactatgca aaaaaa 1319 < 210 > 18 < 211 > 351 < 12 > PRT < 213 > pineapple < 400 > 18 Leu Pro Phe Trp Asn Trp Asp Wing Pro Gly Gly Met Gln He Pro Wing 1 5 10 15 He Tyr Wing Asp Wing Being Ser Pro Leu Tyr Asp Lys Leu Arg Asn Wing 20 25 30 Lys His Gln Pro Pro Thr Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp 35 40 45 Pro Thr Phe Thr Pro Glu Gln Gln He Wing His Asn Leu Thr He Met 50 55 60 Tyr Arg Gln Val He Ser Gly Gly Lys Thr Pro Glu Leu Phe Met Gly 65 70 75"80 Ala Ala Tyr Arg Ala Gly Asp Ala Pro Asp Pro Gly Ala Gly Thr Leu 85 90 95 Glu Leu Val Pro His Asn Thr Met His Leu Trp Thr Gly Asp Pro Asn 100 105 110 Gln Pro Asn Asp Glu Asp Met Gly Thr Phe Tyr Wing Wing Wing Arg Asp 115 120 125 Pro He Phe Phe Wing His His Gly Asn Val Asp Arg Met Trp Tyr Val 130 135 140 Trp Arg Lys Leu Gly Gly Thr His Arg Asp Phe Thr Asp Pro Asp_ Trp Leu Asn Wing Being Phe Leu Phe Tyr Asp Glu Asn Wing Gln Leu Val Arg 165 170 175 Val Lys Val Lys Asp Cys Leu Ser Wing Asp Wing Leu Arg Tyr Thr Tyr 180 185 190 Gln Asp Val Asp He Pro Trp He Ser Wing Lys Pro Thr Pro Lys Lys 195 200 205 Thr Pro Gly Gly Wing Ala Pro Ser Thr Thr Glu Wing He Phe Pro Val 210 215 220 Val Leu Asp Lys Pro Val Ser Ser Thr Val Ala Arg Pro Lys Thr Gly 225 230 235 -240 Arg Ser Thr Gly Glu Glu Glu Val Leu Val Val Glu Gly He Glu Leu 245 250 255 Asp Lys Asp Val Wing Val Lys Phe Asp Val Tyr He Asn Wing Pro Asp 260 265 270 Asn Glu Val Gly Pro Glu Wing Ser Glu Phe Wing Gly Ser Phe Val 275 280 285 Gln Val Pro His Lys His Lys Lys Gly Lys Lys Glu Lys Ala Arg He 290 295 300 Lys Thr Thr Leu Arg Leu Gly He Thr Asp Leu Leu Glu Asp He Gly 305 310 315 320 Wing Glu Asp Asp Glu Ser Val Leu Val Thr Leu Val Pro Arg He Gly 325 330 335 Glu Gly Leu Val Lys Val Gly Gly Leu Arg He Asp Phe Ser Lys 340 345 350 < 210 > 19 < 2X1 > 2181 < 212 > DNA < 213 > pineapple < 220 > < 221 > CDS < 222 > (2) .. (1858) < 400 > 19 c ggt atc gat aag ctt gat cea gtg cct ggt tta ggt gta ttc act atg 49 Gly He Asp Lys Leu Asp Pro Val Gly Leu Gly Val Phe Thr Met 1 5 10 15 gcc acc ctc tet aaa cta gct tc cca c ata ce cct ce cct cc tcc 97 Ala Thr Leu Ser Lys Leu Ala Ser Gln Pro He Thr Pro Pro Leu Ser 20 25 30 ccc cct cct cct cct cct cct cct tct ctc acc aaa age ttc acc acc 145 Pro Leu Pro Pro Leu His Pro Wing Pro Leu Thr Tys Ser Phe Thr Thr 35 40 45 acc ttc ctc tcc cct gta ggg gtc cea aac cac ccc gtc ata aga tet 193 Thr Phe Leu Ser Pro Val Gly Val Pro Asn His Pro Val He Arg Ser 50 55 60 cat gca aat cta agg age aac aag aag ag ccg here age ctg cgg gcc 241 His Ala Asn Leu Arg Be Asn Lys Arg Met Pro Thr Be Leu Arg Wing 65 70 75 80 gca tcg ccc gcc gcg acc tac tcc tgg gcc ctc ggc ggg ctt tac ggt 289 Wing Ser Pro Wing Wing Thr Tyr Ser Trp Wing Leu Gly Gly Leu Tyr Gly 85 90 95 gcc acc act ggg ctc ggc ctc aac cgt gcg gcc gcc gcc gcc cct atc 337 Wing Thr Thr Gly Leu Gly Leu Asn Arg Arg Wing Wing Wing Wing Pro He 100 105 110 ctg gct ccc gac ctc tea act tgt ggg ccg cct gcc gac ctc cct gcc 385 Leu Wing Pro Aap Leu Be Thr Cys Gly Pro Pro Wing Asp Leu Pro Wing 115 120 125 tcc gcc cga ccg here gtt tgc ccg cea tac tcc acc atc atc 433 Ser Ala Arg Pro Thr Val Cys Cys Pro Pro Tyr Gln Ser Thr He He 130 135 140 gac ttc aag ctc ccg ccg cg cg cg cg cg cg c gg ctt cgc ggg cg gcg 481 Asp Phe Lys Leu Pro Pro Arg Ser Wing Pro Leu Arg Val Arg Pro Wing 145 150 155 - -160 gcc cac ttg gt 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 165 170 175 gag ctc atg agg gcc ctg ccg gcc gac gac ccg cgc aac ttc gta cag 577 Glu Leu Met Arg Ala Leu Pro Wing Asp Asp Pro Arg Asn Phe Val Gln 180 185 190 cac gcg aaa gta cac tgt gcg tat tgc gac ggc gcg tat gac ca atc 625 Gln- Ala Lys Val His CyS Wing Tyr Cys Asp Gly Wing Tyr Asp Gln He 195 200 205 ggc ttc ccc gat ctc gag atc cag atc cac aac tcg tgg ctc ttc ttt 673 Gly Phe Pro Asp Leu Glu He Gln He His Asn Ser Trp Leu Phe Phe 210 215 220 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 He Leu Gly Lys 225 230 235 240 ctt atc ggc gac gac acg tcc gcg ctg cct tcc tgg aac tgg gac gcg 769 Leu He Gly Asp Asp Thr Phe Wing Leu Pro Phe Trp Asn Trp Asp Wing 245 250 255 ccg ggg ggc atg cag tcc ccg tet atc tac here gac cct tea tcc tcg 817 Pro Gly Gly Met Gln Phe Pro Ser He Tyr Thr Asp Pro Ser Ser 260 265 270 cta tat gac aag ctg cgt gat gcg aag cac cg ccg gcg act ttg att 865 Leu Tyr Asp Lys Leu Arg Asp Wing Lys His Gln Pro Pro Thr Leu He 275 280 285 gac ctc gac tac aat ggc acc ac ct acc acct tct cct gaa gaa cag 913 Asp Leu Asp Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln 290 295 300 att aac cac aac ctc gcc gtc atg tac cga cag gtg ata tcc agt gga 916 He Asn His Asn Leu Ala Val Met Tyr Arg Gln Val He Ser Ser Gly a? -5 310 315 320 aag acg cea gag ctg ttt atg ggc tea gcg tac cgc gcc ggt gac cag 1009 Lys Thr Pro Glu Leu Phe Met Gly Ser Wing Tyr Arg Wing Gly Asp Gln 325 330 335 cct gac ccc ggc gca ggc tet gta gag cag aag ccg cac ggc ccg gtg 1057 Pr o Asp Pro Gly Wing Gly Ser Val Glu Gln Lys Pro His Gly Pro Val 340 345 350 cat gtg tgg here 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 355 360 365 acg ctc tac tcg gcg gcg tgg gac ccc gtc ttc tcc gca cac gcc 1153 Thr Leu Tyr Ser Wing Wing Trp Asp Pro Val Phe Phe Wing His Gly 370 375 380 aac atc gac cgc atg tgg tae gtg tgg agg aac ctt ggc ggc aag cac 1201 Asn He Asp Arg Met Trp Tyr Val Trp Arg Asn Leu Gly Gly Lys His 385 390 395 400 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 Wing Being Phe Leu Phe Tyr 405 410 415 gat gag aat gcg cag ctc gtc cgt gtt aaa gta aaa gac tgc tta gag 1297 Asp Glu Asn Wing Gln Leu Val Arg Val Lys Val Lys Asp Cys Leu Glu 420 425 430 gcc gac gca atg cgg tac here tac cag gat gata atc ccg tgg ctc 1345 Wing Asp Wing Met Arg Tyr Thr Tyr Gln Asp Val Glu He Pro Trp Leu 435 440 445 aaa gca aag ccg acg cea aag age gcc cta cag aag ata aag age aag 1393 Lys Ala Lys Pro Thr Pro Lys Ser Ala Leu Gln Lys He Lys Ser Lys 450 455 460 gta tcg acg ctg aag gca here cea agg ggg acg acg ac ac ac gca 1441 Val Ser Thr Leu Lyg Wing Thr Pro Arg Gly Thr Thr Thr Thr Thr Wing 465 470 475 480 gag act here ttt ccg gtg gtg ctg gat aag ccg gtg agt gca here gtg 1489 Glu Thr Thr Phe Pro Val Val Leu Asp Lys Pro Val Ser Wing Thr Val 485 490 495 gct aga ccg aag gcc agg agg ggg aag gag aag gaa gag gag 1537 Wing Arg Pro Lys Wing Arg Arg Ser Gly Lys Glu Lys Gl u Glu Glu Glu 500 505 510 gag gtg ttg gtg gtg gag gga atc gag ttg gag aag gac gtg ttc gtg 1585 Glu Val Leu Val Val GLu Gly He Glu Leu Glu Lys Asp Val Phe Val 515 520 525 aag ttt gat gtg tat ata aac tcg ccg gag cac gaa ggg gtg ggg ccg 1633 Lys Phe Asp Val Tyr He Asn Ser Pro Glu His Glu Gly Val Gly Pro - 530 535 540 gag gcg agt gag ttc gca ggg age ttc gtc cac gtg cea cac aag_cac 1681 Glu Ala Ser Glu Phe Ala Gly Ser Phe Val His Val Pro His Lys His 545 550 _ 555 _ 56Q aag aag gcg aag gag aag gag atg gcc agg atg aac agg ctt 1729 Lys Lys Wing Lys Lys Gly Lys Glu Met Wing Arg Met Asn Thr Arg Leu 565 570 575 aag ctc ggg ata acg gac ctg ctc gag gac atc ggc gac gac gac gac 1777 Lys "Leu Gly He Thr Asp Leu Leu Glu Asp He Gly Wing Glu Asp Asp 580 585 590 gag age gtg ctc atc acc ctc gtg ccc agg age ggc aag gga atg gtg 1825 Glu Ser Val Leu He Thr Leu Val Pro Arg Ser Gly Lys Gly Met Val 595 600 605 aag gtt gga ggg cta agg att gat ttc tcc aag tgatgagcat attgtgaaga 1879 Lys Val Gly Gly Leu Arg He Asp Phe Ser Lys 610 615 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 caagttgett ggtgtaatgg 2118 ~ atattcaaca ttgtttccta tacatctttt tttggaagaa aaaaaaaaaa aaaaaaaatc 2178 gat 2181 < 210 > 20 < 211 > 619 < 212 > PRT < 213 > pineapple < 400 > 20 Gly He Asp Lys Leu Asp Pro Pro Val Gly Leu Gly Val Phe Thr Met 1 5 10 15 Wing Thr Leu Ser Lys Leu Wing Pro Gln Pro Pro Th Pro Pro Leu Ser 20 25 30 Pro Leu Pro Pro Leu Pro Pro Ser Leu Thr Lys Ser Phe Thr Thr 35 40 45 Thr Phe Leu Ser Pro Val Gly Val Pro Asn His Pro Val He Arg Ser 50 55 60 His Wing Asn Leu Arg Ser Asn Lys Arg Met Pro Thr Ser Leu Arg Wing 65 70 75 80 Wing Ser Pro Wing Wing Thr Tyr Wing Trp Wing Leu Gly Gly Leu Tyr Gly 85 90 95 Wing Thr Thr Gly Leu Gly Leu Asn Arg Wing Wing Wing Wing Pro Pro 100 105 110 Leu Wing Pro Asp Leu Being Thr Cys Gly Pro Pro Wing Asp Leu Pro Wing 115 120 12S Wing Wing Arg Pro Thr Val Cys Pro Pro Tyr Gln Ser Thr He He 130 135 140 _- Asp Phe Lys Leu Pro Pro Arg Ser Wing Pro Leu Arg Val Arg Pro Wing 145 150 155 160 Wing His Leu Val Asp Wing Asp Tyr Leu Wing Lys Tyr Lys Lys Wing Val 165 170 175 Glu Leu Met Arg Wing Leu Pro Wing Asp Asp Pro Arg Asn Phe Val Gln 180 185 190 Gln Ala Lys Val His Cys Wing Tyr Cys Asp Gly Wing Tyr Asp Gln He 195 200 2O5 Gly Phe Pro Asp Leu Glu He Gln He His Asn Ser Trp Leu Phe Phe 210 215 220 Pro Trp His Arg Phe Tyr Leu Tyr Ser Asn Glu Arg He Leu Gly Lys 225 230 235 -240 Leur He Gly Asp Asp Thr Phe Wing Leu Pro Phe Trp Asn Trp Asp Wing 245 250 255 Pro Gly Gly Met Gln Phe Pro Be He Tyr Thr Asp Pro Be Ser 260 265 270 Leu Tyr Asp Lys Leu Arg Asp Wing Lys His Gln Pro Pro Thr Leu He 275 280 285 Asp Leu Asp Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Glu Glu Gln 290 295 300 He Asn His Asn Leu Wing Val Met Tyr Arg Gln Val He Ser Ser Gly 305 310 313 _ 320 Lys Thr Pro Glu Leu Phe Met Gly Ser Wing Tyr Arg Wing Gly Asp Gln 325 330 335 Pro Asp Pro Gly Wing Gly Ser Val Glu Gln Lys Pro His Gly Pro Val 340 345 350 His Val Trp Thr Gly Asp Arg Asn Gln Pro Asn Arg Glu Asp Met Gly 355 360 365 Thr Leu Tyr Ser Ala Ala Trp Asp Pro Val Phe Phe Ala His His Gly 370 375 380 Asn He Asp Arg Met Trp Tyr Val Trp Arg Asn Leu Gly Gly Lys His 385 390 395 400 Arg Asn Phe Thr Asp Pro Asp Trp Leu Asn Wing Being Phe Leu Phe Tyr 405 410 415 Asp Glu Asn Wing Gln Leu Val Arg Val Lys Val Lys Asp Cys Leu Glu 420 425 430 Wing Asp Wing Arg Tyr Thr Tyr Gln Asp Val Glu He Pro Trp Leu 435 440 445 Lys Wing Lys Pro Thr Pro Lys Ser Wing Leu Gln Lys He Lys Ser Lys 450 455 460 Val Ser Thr Leu Lys Wing Thr Pro Arg Gly Thr Thr Thr Thr Thr Wing 465 470 475 480 Glu Thr Thr Phe Pro Val Val Leu Asp Lys Pro Val Ser Wing Thr Val 485 - 490 495 Wing Arg Pro Lys Wing Arg Arg Ser Gly Lys Glu Lys Glu Glu Glu Glu 500 505 510 Glu Val Leu Val Val Glu Gly He Glu Leu Glu Lys Asp Val Phe Val 515 520 525 Lys Phe Asp Val Tyr He Asn Ser Pro Glu His Glu Gly Val Gly Pro 530 535 540 Glu Wing Ser Glu Phe Wing Gly Ser Phe Val His Val Pro His Lys His 545 550 555 560 Lys "Lys Wing Lys Lys Gly Lys Glu Met Wing Arg Met Asn Thr Arg Leu 565 570 575 Lys Leu Gly He Thr Asp Leu Leu Glu Asp He Gly Wing Glu Asp Asp 580 585 590 Glu Ser Val Leu He Thr Leu Val Pro Arg Ser Gly Lys Gly Met Val 595 600 605 Lys Val Gly Gly Leu Arg He Asp Phe Ser Lys 610 615 < 210 > 21 < 211 > 22 < 212 > DNA < 213 > Artificial sequence < 220 > < 22 > Description of the artificial sequence: initiator < 400 > 21 gcctgcagtt ytcrtcrtag aa < 210 > 22 < 211 > 28 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 22 gcgaattcga tccnacntty gckttncc < 210 > 23 < 211 > 24 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 23 gcgaattctn caytgygcnt aytg < 210 > 24 < 211 > 27 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 24 gcgaattctt nccntwytgg aaytggg < 210 > 25 < 211 > 26 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 25 gcctgcagcc acatnckrtc nacrtt < 210 > 26 < 211 > 22 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 4O0 > 26 gcctgcagtt ytcrtcrtag aa < 210 > 27 < 211 > 25 < 212 > DNA < 213 > Artificial sequence < 22XL > < 223 > Description of the artificial sequence: initiator < 400 > 27 gttgctcttc ttaggctcgg cttac < 210 > 28 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 28 gactcgagtc gacatcga < 210 > 29 < 211 > 25 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 29 atatcacctg tcggtacatg acggc < 210 > 30 < 211 > 25 < 212 > DNA < 213 > Artificial sequence < 22) > < 223 > Description of the artificial sequence: initiator < 400 > 30 gtgccattgt agtcgaggtc aatca < 2U0 > 31 < 2H > 26 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator- < 400 > 31 ccagtgcctg gtttaggtgt attcac < 210 > 32 < 211 > 35 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 32 gactcgagtc gacatcgatt tttttttttt ttttt < 210 > 33 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223- > Description of the artificial sequence: initiator < 400 > 33 gactcgagtc gacatcga

Claims (22)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as novelty and therefore the content of the following claims is claimed as property. An isolated nucleic acid molecule characterized in that it comprises a nucleotide sequence that encodes or is complementary to a nucleotide sequence encoding a banana, tobacco or pineapple PPO polypeptide having an amino acid sequence set forth in any of SEQ ID US: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20, or comprising the copper binding site of any of the amino acid sequences.
2. 2. An isolated nucleic acid molecule encoding a banana, tobacco or pineapple PPO polypeptide, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence set forth in any 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 sequence of nucleotides of (i) or (ii); and (iv) a nucleotide sequence that is complementary to 8i) or (ii) or (iii).
3. 3. The isolated nucleotide sequence according to claim 1 or 2, characterized in that the copper binding site is the CuA binding site of the banana, tobacco or pineapple PPO polypeptide.
4. 4. The isolated nucleotide sequence according to claim 1 or 2, characterized in that the copper binding site is the CuB binding site of the banana, tobacco or pineapple PPO polypeptide.
5. 5. The isolated nucleic acid molecule according to any of claims 1 to 4, characterized in that the banana PPO polypeptide is at least expressed in the shell of the banana.
6. 6. The isolated nucleic acid molecule according to any of claims 1 to 4, characterized in that the tobacco PPO polypeptide is at least expressed in tobacco leaves.
7. 7. The isolated nucleic acid molecule according to any of claims 1 to 4, characterized in that the pineapple PPO polypeptide is at least expressed in pineapple fruit.
8. 8. A recombinant vector characterized in that it comprises the isolated nucleic acid molecule according to any of claims 1 to 7 inserted into a vector molecule.
9. 9. The recombinant vector according to claim 8, characterized in that the vector is a plasmid expression vector.
10. 10. The recombinant vector according to claim 9, characterized in that the plasmid expression vector is Bluescript SK *.
11. 11. The recombinant vector according to claim 8, characterized in that the vector is a binary vector suitable for introducing it into a cell, tissue or organ of a plant.
12. 12. The recombinant vector according to any of claims 8 to 11, characterized in that the vector is capable of being replicated, and the nucleotide sequence encoding the PPO is capable of being transcribed and translocated into a unicellular organism or into a plant .
13. 13. A method for increasing the activity level of banana, pineapple or tobacco PPO in a plant or a cell, tissue or organ thereof, the method comprises: (i) introducing a nucleotide sequence to the plant or a cell, tissue or organ thereof, the sequence encodes a polypeptide of Banana, tobacco or pineapple PPO having an amino acid sequence set forth in any 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 of the amino acid sequences; and (ü) expressing the nucleotide sequence to produce an enzymatically active PPO polypeptide.
14. 14. A method for increasing the activity level of banana, pineapple or tobacco PPO in a plant or a cell, tissue or organ thereof, the method comprising: (i) introducing a nucleic acid molecule into the plant or a cell, tissue or organ thereof, the nucleic acid molecule comprises the nucleotide sequence set out in any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19, or a degenerate sequence of nucleotides thereof; and (ii) expressing the nucleic acid molecule to produce an enzymatically active PPO polypeptide.
15. 15. A method for lowering the level of activity of the PPO in a plant or a cell, tissue or organ thereof, the method is characterized in that it comprises introducing a nucleic acid molecule to the plant or a cell, tissue or organ of the same comprising a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence encoding a banana, tobacco or pineapple PPO polypeptide having an amino acid sequence set forth in any of SEQ ID NOS: 2 , 4, 6, 8, 10, 12, 14, 16, 18 or 20, or the copper binding site of any of the amino acid sequences; (ii) a nucleotide sequence set forth in any 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. 16. The method according to claim 15, characterized in that it additionally comprises expressing the introduced nucleic acid molecule to produce direct or antisense RNA therefrom.
17. 17. The method according to claim 15 or 16, characterized in that the activity of the PPO is decreased by co-suppression of genes encoding endogenous PPO, which would otherwise be expressed in the plant or a cell, tissue or organ of the same.
18. 18. The method according to claim 15 or 16, characterized in that the activity of the PPO is decreased by the expression of antisense RNA which is complementary to the RNA encoded by a gene encoding endogenous PPO that would otherwise be expressed in the plant or a cell, tissue or organ thereof.
19. 19. The method according to any of claims 13 to 18, characterized in that the nucleic acid molecule is introduced into the plant or a cell, tissue or organ thereof by means of transformation mediated by Agroba. c ter i um
20. 20. The method according to any of claims 13 to 18, characterized in that the nucleic acid molecule is introduced into the plant or a cell, tissue or organ thereof by means of microparticle bombardment using an acid-coated microproject. nucleic.
21. 21. A transformed plant characterized in that it comprises the isolated nucleic acid molecule according to any of claims 1 to 7, or a part of the plant, progeny or propagule thereof, which also comprises the nucleic acid molecule.
22. 22. A transformed plant characterized in that it comprises the recombinant vector according to any of claims 8, 9, 11 or 12, or a part of the plant, progeny or propagule thereof, which also comprises the leico.