Polyphenol oxidase genes

Details Polyphenol oxidase genes Polyphenol oxidase genes

1995-06-07

2004-03-09

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide encodes an inhibitory rna molecule

C800S298000, C435S252300, C435S320100, C435S468000, C536S025100

Reexamination Certificate

active

06703542

ABSTRACT:

The present invention relates to a method of modifying polyphenol oxidase (PPO) activity in fruit and vegetables and to DNA sequences for use therein.
Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products. Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.
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.
It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed. If the amount of this enzyme could be decreased the susceptibility of the tissue to brown would be reduced.
PPO sequence information may be used to construct synthetic genes which genes may be transformed into plants to decrease expression of the normal PPO gene, thereby decreasing synthesis of the enzyme.
It will also be understood that in certain instances the browning reactions in plants are desirable, such as in the production of black tea, cocoa, coffee, black pepper, black olives, etc. In these instances it may be desirable to increase the level of PPO to produce desired levels of browning or changes in flavour compounds.
The role of PPO in normal plant growth and development is not understood at present. There are a number of instances where increased levels of this enzyme are correlated with increased resistance to plant pathogens. It follow that genetic manipulation of plants to increase the level of PPO activity may confer useful resistance against pathogens and pests.
The grapevine PPO gene codes for an additional 103 amino acids upstream of the N-terminus of the mature protein. This region has the properties of a chloroplast transit peptide and is most likely responsible for targeting of the protein to be imported into the chloroplast and processed to produce the mature PPO protein. Transformation of plants with this gene may therefore result in correct targeting and maturation of the grapevine PPO in other species and result in accumulation of active grapevine PPO enzyme in the plastids of these tissues.
The terms “gene encoding PPO”, “gene coding for PPO” or “PPO gene” as used herein should be understood to refer to the PPO gene or a sequence substantially homologous therewith.
In a first aspect of the present invention, there is provided a DNA sequence including a gene coding for a polypeptide having plant polyphenol oxidase (PPO) activity or a fragment thereof.
The DNA sequence may include a pre-sequence of a plant PPO gene coding for a transit peptide.
The DNA sequence may be modified. The DNA sequence may include a sequence coding for antisense mRNA to a plant PPO gene, or a fragment thereof.
The DNA sequence may include a catalytic cleavage site.
Alternatively the presequence may be replaced by other targeting sequences to direct the polypeptide having plant PPO activity to other cellular compartments.
The DNA sequence may include a putative chloroplast transit sequence and a mature grape vine PPO protein, as illustrated in FIG.
1
.
The DNA sequence may include a gene coding for a polypeptide having a broad bean leaf PPO activity, as illustrated in FIG.
2
.
The DNA sequence may include a gene coding for a polypeptide having apple fruit PPO activity, as illustrated in FIG.
3
.
The DNA sequence may include a gene coding for a polypeptide having potato tuber PPO activity, as illustrated in FIG.
4
.
In a further aspect of the present invention there is provided a DNA sequence including a sequence coding for antisense mRNA to a plant PPO gene, or a fragment thereof.
In a further aspect of the present invention there is provided a method for preparing a recombinant DNA plasmid including a DNA sequence coding for a polypeptide having plant PPO activity or a fragment thereof, which method includes
providing
a DNA sequence including a gene coding for a polypeptide having PPO activity or a fragment thereof; and
a plasmid expression vector; and
reacting the DNA sequence and the plasmid expression vector to deploy the DNA sequence within the plasmid expression vector.
The DNA sequence coding for PPO may be formed from polyadenylated RNA, for example isolated from a plant sample. The plant may be selected from apples, potatoes, grapes and beans. Preferably the plant sample is isolated from sultana grape berries, broad bean leaves, apple peel or cortex or potato tubers.
In order to provide a DNA sequence coding for PPO, in a preferred aspect of the present invention the method for the preparation of a recombinant DNA plasmid may include the preliminary steps of
providing a source of a polypeptide having plant PPO activity;
isolating polyadenylated RNA coding for a polypeptide having plant PPO activity therefrom; and
treating the polyadenylated RNA to construct copy DNA (cDNA).
The isolation of the polyadenylated RNA may be conducted utilising an oligo-dT spun column.
The step of treating the polyadenylated RNA to construct cDNA according to this aspect of the present invention may include
treating the polyadenylated RNA with reverse transcriptase and an adapter primer to form first strand cDNA; and
amplifying the cDNA so formed using the polymerase chain reaction (PCR).
The step of reacting the polyadenylated RNA with reverse transcriptase may utilise an oligonucleotide adapter primer having the sequence
5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT(SEQ ID NO: 15)
The step of amplifying the cDNA may utilise an adapter primer having the sequence
5′-GACTCGAGTCGACATCG(SEQ ID NO: 16) and a 5′-end primer.
The 5′-end primer may have the sequence
5′-CCIATICAGGCICCIGATATIICIAAGTGTGG(SEQ ID NO: 17) when utilized for the amplification of grape cDNA.
The 5′-end primer may have the sequence
5′-GCGGATCCTT[CT]TA[CT]GA[CT]GA[GA]AA[CT]AA(SEQ ID NO: 18).
when utilized for the amplification of bean cDNA.
The 5′-end primer may have the sequence
5′-GCGAATTCGA[AG]GA[TC]ATGGGIAA[TC]TT[TC]TA)(SEQ ID NO: 19)
when utilised for the amplification of apple cDNA.
The 5′-end primer may have the sequences
GEN3: (5′-GCGAATTCTT[TC][TC]TICCITT[TC]CA[TC][AC]G)(SEQ ID NO: 20)
GEN7: (5′-GCGAATTCAA[TC]GTIGA[TC][AC]GIATGTGG)(SEQ ID NO: 21)
when utilised for the amplification of potato cDNA.
Alternatively, the step of treating the polyadenylated RNA to construct cDNA according to this aspect of the present invention may include
treating the polyadenylated RNA with reverse transcriptase and a PPO specific primer to form first strand cDNA;
treating the cDNA so formed with terminal d Transferase to attach a polyadenosine tail sequence at the 3′ end of the cDNA; and
amplifying the polyadenylated cDNA so formed by PCR.
The step of treating the polyadenylated RNA with reverse transcriptase may utilise a PPO specific oligonucleotide primer having the sequence
5′-AATCTTTGTGGTGACTGGCG(SEQ ID NO: 22)
for grape PPO or the PPO-specific primer is an oligonucleotide primer having the sequence
5′-GACGGTACATTAGTCTTAAAT(SEQ ID NO: 23)
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