Inducibile plant promoters

Details Inducibile plant promoters Inducibile plant promoters

1999-10-05

2004-02-17

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide contains a tissue, organ, or cell...

C435S320100, C435S468000, C435S419000, C435S006120, C536S024100, C800S315000, C800S286000, C800S283000, C800S279000, C800S302000

Reexamination Certificate

active

06693227

ABSTRACT:

TECHNICAL FIELD
The present invention relates to polynucleotides which may be useful in recombinant plant DNA technology or analysis, in particular to tissue- or ripening-specific promoter DNA, and products and methods employing such DNA.
BACKGROUND ART
It is desirable to be able to specifically express (or inhibit the expression of) genes in plants, for instance in particular tissues, or at a particular developmental stage. This may allow particular biosynthetic enzymes to be produced only in the fruit of a plant, and not in other tissues wherein it may have undesirable effects. Likewise it may be desirable to have particular protective proteins (e.g. anti-fungal, pesticidal) expressed only during a particular vulnerable developmental stage e.g. early or late ripening.
This type of specific expression can be achieved by using inducible promoters which are ‘switched on’ in the presence of environmental signals present only in restricted tissues of the plant, or only at particular times. Such promoters have already been made available for tomatoes. Thus WO93/07257 (SPI Inc.) relates, inter alia, to gene-fusions capable of conferring tissue-specific or developmentally regulated gene constructs. These constructs apparently allow particular genes to be expressed during the formation and ripening of fruit. The coding region of clone &lgr;UC82-3.3 in WO93/07257, which was derived from tomato, has homology to a bacterial histidine decarboxylase (HDC). Similarly WO94/13797 (CSIRO) relates, inter alia, to inducible soft-fruit promoter DNA derived from alcohol dehydrogenase (ADH) in tomatoes. ADH apparently has a role in ripening in that it metabolises alcohols and aldehydes involved in flavour. The ADH promoter is apparently sensitive to and therefore inducible by high levels of O
2
.
It is clear from the foregoing that the disclosure of novel inducible promoters, particularly those active in plants other than tomato plants, would provide a useful contribution to the art.
The applicants have now isolated inducible promoters from apple, elements of which show useful properties and which may be useful in particular in the isolation of other ripening specific promoters or transcription factors, or in the genome mapping studies.
DISCLOSURE OF THE INVENTION
In a first aspect of the present invention there is disclosed a recombinant polynucleotide comprising a promoter sequence being: (a) an inducible promoter obtainable from apple, or (b) a functional portion therof, or (c) a functional derivative or homolog promoter being at least 70% homologous to either.
As used herein, “promoter” refers to a non-coding region of DNA involved in binding of RNA polymerase and other factors that initiate or modulate transcription whereby an RNA transcript is produced. Promoters, depending upon the nature of the regulation, may be constitutive or inducible. A constitutive promoter is always turned on. An inducible promoter requires specific signals in order for it to be turned on or off. These may be particular signals for example chemical signals, which are applied to a cell under certain conditions or as a result of a deliberate application. In the context of the present application, the term “inducible” is intended to include particularly promoters which are tissue-specific in that they are effective only in certain plant tissues either with or without externally applied inducing agents, or ripening specific promoters which switched on within some or all plant cells as a result of ripening, for example in response to ethylene produced during the ripening process.
Examples of promoters of the invention include a ABG1 &bgr;-galactosidase promoter whose sequence is included within the sequence shown in
FIG. 3
hereinafter (SEQ ID NO 1); and the ACC synthase promoter whose sequence is comprised within the sequence shown in
FIG. 5
(SEQ ID NO 2) hereinafter.
Thus, the invention provides a promoter comprising at least a functional portion of the Sequence shown in
FIG. 3
or FIG.
5
.
As well as authentic promoters obtainable from apple, the invention also embraces functional portions thereof.
The term “functional” is used herein to describe moieties which have the activity of a promoter as defined above, when present in apple cells.
Also embraced by present invention are functional derivative promoters being at least 70% homologous to the above.
By “derivative” is meant a sequence may be obtained by introducing changes into the full-length or part length sequence, for example substitutions, insertions, and/or deletions. This may be achieved by any appropriate technique, including restriction of the sequence with an endonuclease followed by the insertion of a selected base sequence (using linkers if required) and ligation. Also possible is PCR-mediated mutagenesis using mutant primers. Such changes may be introduced e.g. to remove or incorporate restriction sites into the sequence.
Also embraced by the present invention are functional “homologs” of authentic promoters obtained from apple which hybridise thereto and are at least 70% homologous to either the full-length or part length sequences and in particular to SEQ ID NOS 1 and 2 identified herein.
Such homologs may conveniently be identified and isolated by those skilled in the art from a test sample as follows:
The test sample is contacted with the apple promoter under suitable hybridisation conditions, and any test DNA (e.g. an apple genomic library) which hybridises thereto is identified.
Such screening is initially carried out under low-stringency conditions, which comprise a temperature of about 37° C. or less, a formamide concentration of less than about 50%; and a moderate to low salt (e.g. Standard Saline Citrate (‘SSC’)=0.15 M sodium chloride; 0.15 M sodium citrate; pH 7) concentration. Alternatively, a temperature of about 50° C. or less and a high salt (e.g. ‘SSPE’=0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferably the screening is carried out at about 37° C., a formamide concentration of about 20%, and a salt concentration of about 5×SSC, or a temperature of about 50° C. and a salt concentration of about 2×SSPE. These conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect homology for the identification of a stable hybrid. The phrase ‘substantial similarity’ refers to sequences which share at least 50% overall sequence identity. Preferably, hybridisation conditions will be selected which allow the identification of sequences having at least 70k sequence identity with the probe, while discriminating against sequences which have a lower level of sequence identity with respect to the probe.
After low stringency hybridization has been used to identify one or more homologs having a substantial degree of similarity with the probe sequence, this subset is then subjected to high stringency hybridization, so as to identify those clones having a particularly high level of homology with respect to the probe sequences. High stringency conditions comprise a temperature of about 42C or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration. Alternatively they may comprise a temperature of about 65C or less, and a low salt (SSPE) concentration. Preferred conditions for such screening comprise a temperature of about 42C, a formamide concentration of about 20%, and a salt concentration of about 2×SSC, or a temperature of about 65C, and a salt concentration of about 0.2 SSPE.
Thus, according to the present invention the derivative sequence or homolog is at least 70% identical to the sequence of the full or part-length promoters. Typically there is 80% or more, 90% or more 95% or more or 98% or more identity between the derivative or homolog and the authentic sequences. There may be up to five, for example up to ten or up to twenty nucleotide deletions, insertions and/or substitutions made to the full-length or part leng

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