Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-08-04
2001-06-05
Nelson, Amy J. (Department: 1638)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091400, C435S320100, C536S024300
Reexamination Certificate
active
06242221
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to the isolation of genes encoding polyphenol oxidase (PPO) from plants. More particularly, the present invention provides methods for obtaining genomic DNA encoding PPO enzymes and fragments of a wide range of plants, such as, for example, strawberry, tobacco, apricot, avocado, cherry, peach, pear, coffee, apple, lettuce, French bean, banana, rice, and potato. The present invention clearly extends to the isolated genomic DNAs encoding said PPO enzymes and fragments.
Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products. Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.
It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed.
It will be apparent from the preceding discussion that there is a need to develop methods for reducing the level of browning of plant tissues. One approach is to modulate the level of expression of PPO genes in plants. A prerequisite to achieving this objective by molecular means is the isolation of nucleic acid encoding PPO genes from a variety of plant sources.
2. Description of Related Art
The prior art includes International Application PCT/AU92/00356 to the present applicant which describes the cloning of PPO genes from grapevine, broad bean leaf, apple fruit and potato tuber. This application recognises that PPO levels in plants may be manipulated by increasing or decreasing expression of PPO gene. The application also identifies two conserved copper binding sites in PPO genes, designated CuA and CuB, and predicts that these regions are suitable for design of probes and primers to obtain other plant PPO genes. However, the method described in PCT/AU92/00356 suffers from the disadvantages that it is necessary to identify the appropriate stage of development of the target tissue, isolate mRNA or total RNA and synthesize cDNA. This in turn requires a relatively large amount of plant material.
SUMMARY OF THE INVENTION
This application is derived from International Patent Application No. PCT/AU97/00041 filed on Jan. 24, 1997, which claims Paris Convention priority from Australian Patent Application No. PO 7856 filed on Feb. 5, 1996, and from Australian Patent Application No. PO 2361 filed on Sep. 16, 1996, the entire contents of which are incorporated herein by way of reference.
Bibliographic details of the publications referred to in this specification by author are collected at the end of the description.
Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification appear after the claims.
Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
In work leading up to the present invention, the inventors sought to produce improved methods for isolating PPO-encoding nucleic acid molecules which are susceptible for use in modifying the expression of endogenous PPO genes in plants, to reduce browning and modify ripening and storage characteristics of plant tissues and organs.
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.
The inventors have cloned several PPO-encoding genomic DNAs from strawberry, tobacco, apricot, avocado, cherry, peach, pear, coffee, apple, lettuce, French bean, banana, rice, and potato and produced recombinant gene constructs comprising same for the expression of recombinant PPO polypeptides and nucleic acids capable of modifying the PPO content of plant tissues and cells when expressed therein.
Accordingly, a first aspect of the present invention provides a method for preparing nucleic acid encoding PPO, fragments and derivatives thereof, which method includes the steps of:
(a) providing:
(i) a sample of plant tissue,
(ii) a first primer in sense orientation having a sequence corresponding to a conserved region of a PPO gene,
(iii) a second primer in antisense orientation having a sequence corresponding to a conserved region of a PPO gene;
(b) isolating genomic DNA from said plant tissue; and
(c) amplifying the genomic DNA using the first and second primers.
Preferably, the conserved region of a PPO gene is contained within or is in close proximity to a nucleotide sequence of a PPO gene that encodes the CuA or CuB region of a PPO polypeptide.
In a further aspect of the present invention, there is provided isolated nucleic acid comprising a nucleotide sequence which encodes PPO or a complementary nucleotide sequence thereto, or a fragment or derivative of said nucleic acid, wherein said nucleic acid os derived from a plant selected from the group consisting of strawberrry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip, and wheat. This aspect of the invention clearly extends to any nucleic acid isolated using the inventive method described herein. Preferably, the nucleic acid comprises genomic DNA or an equivalent thereof which lacks introns.
A further aspect of the present invention provides a method for preparing a recombinant vector which includes nucleic acid comprising a nucleotide sequence encoding PPO or complementary thereto, or a fragment or derivative of said nucleic acid, wherein said nucleic acid is derived from a plant selected from the group consisting of strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip and wheat, and wherein said method includes:
(i) providing said nucleic acid and a vector; and
(ii) reacting said nucleic acid and said vector to deploy the nucleic acid within the vector.
A further aspect of the present invention provides a recombinant vector capable of being replicated, transcribed and translated in a unicellular organism or a plant, wherein said vector includes isolated nucleic acid comprising a nucleotide sequence encoding PPO or a complementary nucleotide sequence thereto, or a fragment or derivative of said nucleic acid, wherein said nucleic acid is derived from a plant selected from the group consisting of strawberry, tobacco, apricot, avocado, cherry, peach, pear, pineapple, tea, coffee, apple, lettuce, French bean, banana, rice, potato, parsnip and wheat.
Commonwealth Scientific and Industrial Research Organization
Cooper & Dunham LLP
Nelson Amy J.
White John P.
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