Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-12-13
2002-09-24
Fredman, Jeffrey (Department: 1656)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S320100
Reexamination Certificate
active
06455675
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the regulation of plant growth, and more particularly to the molecular cloning and expression of a gibberellin 20-oxidase gene and its use, for example in transgenic plants.
2. Description of the Related Art
Chemical compounds for control of plant growth have been in commercial use for many years. Many of these compounds act by inhibiting various steps in the biosynthesis of gibberellins (GAs). GAs form a large group of diterpenoid natural products, some members of which function as hormones in plants, controlling many aspects of development, including, for example, shoot elongation. Among the groups of compounds which compounds, compounds with a nitrogen-containing heterocycle, and acylcyclohexanediones. However, the use of such chemicals involves several problems. It is, for example, difficult to apply the chemicals to plants in the appropriate quantities, or to select plant organs, without the chemicals spreading to other plants or animal life. There is a risk of persistence which can make it difficult to grow other crops subsequently to treated crops. A problem addressed by the present invention is therefore to avoid the use of such chemicals. This problem can be solved within this application by providing means for plant growth control at the plant gene level.
The later steps of the GA biosynthetic pathway are catalysed by soluble 2-oxoglutarate-dependent dioxygenases, several of which have been proposed as regulatory enzymes in the biosynthesis of the physiologically important C
19
compound, GA
1
. For example, the activity of the GA 20-oxidase is enhanced by long days in certain photoperiod-sensitive plants and is down-regulated as a consequence of GA
1
action in several species.
SUMMARY OF THE INVENTION
According to the invention, there is provided a DNA sequence which encodes a polypeptide exhibiting GA 20-oxidase activity. This disclosure is the first example of the molecular cloning of a GA:2-oxoglutarate dioxygenase. The enzyme GA 20-oxidase is also known as a 20-hydroxylase or C-20 oxidase, as it catalyses oxidation reactions at the C-20 carbon atom of the GA structure. It is a dioxygenase, as oxoglutarate is simultaneously oxidised.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As demonstrated in the Examples the DNA sequence of the present invention encodes GA 20-oxidase capable of acting essentially on one or more of the following substrates: GA
12
, GA
53
, GA
15
(open or closed lactone), GA
44
(open or closed lactone), GA
24
, GA
19
and GA
23
among others.
The present invention thus further relates to a DNA sequence encoding a polypeptide exhibiting GA 20-oxidase activity, in which the polypeptide exhibiting GA 20-oxidase activity is capable of acting essentially on one or more of the following substrates: GA
12
, GA
53
, GA
15
(open or closed lactone), GA
44
(open or closed lactone), GA
24
, GA
19
and GA
23
The DNA sequence of the invention may encode a GA 20-oxidase from, in principle, any plants or fungi, but preferably from monocotyledonous and dicotyledonous plants, and more preferably from dicotyledonous plants. A particularly suitable source is plants of the family Cucurbitaceae, such as
C. maxima
, of which the immature seeds are a convenient source. A further suitable source is plants of the family Cruciferae, such as
Arabidopsis thaliana
, of which shoot material is a convenient source.
A preferred embodiment of the invention is therefore a DNA sequence which encodes a GA 20-oxidase obtainable from plants or fungi, preferably from monocotyledonous and dicotyledonous plants respectively, more preferably from dicotyledonous plants and most preferably from plants of the family Cucurbitaceae and Cruciferae respectively, such as
C. maxima
and
Arabidopsis thaliana
, or a protein having substantial homology thereto.
As used in the present application, substantial sequence homology means close structural relationship between sequences of nucleotides or amino acids. For example, substantially homologous DNA sequences may be 60% homologous, preferably 80% and most preferably 90% or 95% homologous, or more, and substantially homologous amino acid sequences may preferably be 35%, more preferably 50%, most preferably more than 50% homologous. Homology also includes a relationship wherein one or several subsequences of nucleotides or amino acids are missing, or subsequences with additional nucleotides or amino acids are interdispersed.
The term “homology” as used herein not only embraces structural homology but also functional homology.
The invention thus further relates to a DNA sequence, which encodes a GA 20-oxidase obtainable from
Cucurbita maxima
or
Arabidopsis thaliana
or a protein having at least 35%, preferably at least 50%, and most preferably more than 50% homology therewith.
More specifically, the invention relates to a DNA having a sequence corresponding to the open reading frame of the sequence shown in SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5, or an equivalent sequence through the degeneracy of the genetic code, including derivatives capable of hybridizing with the sequence shown in SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 5, and still encoding a polypeptide exhibiting GA 20-oxidase activity.
A preferred embodiment of the invention is therefore a substantially pure DNA as shown in SEQ ID NO 1, SEQ ID NO 3, or SEQ ID NO 5, or having substantial sequence homology to the sequence shown in SEQ ID NO 1, SEQ ID NO 3, or SEQ ID NO 5.
The DNA sequence according to the invention is preferably a recombinant DNA comprising a DNA sequence which encodes a recombinant polypeptide exhibiting GA 20-oxidase activity. In one embodiment of the invention the recombinant DNA is in the form of a cDNA clone.
It is a further object of the invention to provide a chimaeric gene construct comprising a DNA sequence encoding a polypeptide exhibiting GA 20-oxidase activity in operable linkage with plant expression signals including promoter and termination sequences capable of causing the gene to express a polypeptide exhibiting GA 20-oxidase activity within a plant, wherein the promoter sequences are preferably those of an inducible promoter or a tissue-preferential or a tissue-specific promoter.
The invention further comprises a chimaeric gene construct comprising at least a part of a reverse GA 20-oxidase nucleotide sequence, in operable linkage with plant expression signals including promoter and termination sequences capable of causing the reverse sequence to express antisense mRNA within a plant.
It is also an object of the invention to provide transformed host cells comprising recombinant DNA encoding a polypeptide exhibiting GA 20-oxidase activity in operable linkage with expression signals including promoter and termination sequences which permit expression of said DNA in the host cell.
A preferred embodiment of the invention is a transgenic plant including seed and progeny or propagules thereof comprising preferably stably integrated into its genome a chimeric gene construct as mentioned hereinbefore. Preferred is a monocotyledonous and a dicotyledonous plant, respectively such as tobacco, tomato, cotton, sunflower, maize, wheat and
Dactylis glomerata.
Especially preferred is a transgenic plant which is a monocotyledonous plant, preferably a maize plant or a wheat plant
The invention also comprises a recombinant polypeptide obtainable from plants or fungi exhibiting GA 20-oxidase activity, which polypeptide is preferably capable of acting essentially on one or more of the following substrates: GA
12
, GA
53
, GA
15
(open or closed lactone), GA
44
(open or closed lactone), GA
24
, GA
19
and GA
23
.
A preferred embodiment of the invention is therefore a recombinant polypeptide which exhibits a GA 20-oxidase activity and which is obtainable from plants or fungi, preferably from monocotyledonous and dicotyledonous plants, respectively, more preferably from dicotyledonous plants and most preferably from plants of the family Cucurbitaceae and Cruciferae respectively, such as
C.
Graebe Jan E.
Hedden Peter
Lange Theodor
Phillips Andrew
Chunduru Suryaprabha
Fredman Jeffrey
Long Ashton Research Station
Wenderoth , Lind & Ponack, L.L.P.
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