Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
1999-12-03
2002-04-30
Sisson, Bradley L. (Department: 1655)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C800S295000, C536S023100, C536S024100, C435S468000, C435S410000, C435S419000
Reexamination Certificate
active
06380464
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding a flavonol biosynthetic enzyme in plants and seeds.
BACKGROUND OF THE INVENTION
Flavonoids, of which anthocyanins are the most abundant in plants, represent a large class of molecules that serve many diverse functions, such as co-pigments in flower color, stimulation of pollen tube growth, pollinator attraction, and feeding deterrents and protection against UV irradiation in fruits and seeds (Holton, T. A. et al. (1993)
Plant J
4(6):1003-1010). Therefore, there has been considerable interest in understanding flavonoid biosynthesis, which in tarn would afford the ability to control flower color and other phenotypic traits associated with flavonoid synthesis. Flavonol synthase (FLS) is an enzyme involved in flavonol biosynthesis. This enzyme catalyses the conversion of the dihydroflavonols, dihydrokaempferol and dihydroquercetin to the flavonols kaempferol and quercetin. Dihydroflavonols are the precursors of both anthocyanin and flavonol biosynthesis. In most plants anthocyanins and flavonols are synthesized within the same cell and usually accumulate in the same subcellular location (Holton, T. A. et al. (1995)
Plant cell
7:1071-1083). By modulating the flux of dihyroflavonols through the anthocyanin and flavonol pathways it may be possible to alter phenotypic traits associated with these pathways. For example if the activity of FLS is inhibited, more dihydroflavonol precursors may be available to the enzymes involved in anthocyanin synthesis. It has been shown in petunia that if an enzyme in the anthocyanin pathway (flavonoid 3′5′-hydroxylase) is inactivated, flavonols accumulate due to in part to FLS activity (Holton, T. A. et al. (1995)
Plant Cell
7:1071-1083). Thus the accumulation of specific flavonoids is in part due to the activity of enzymes in the anthocyanin and flavonol biosynthetic pathways.
There is a great deal of interest in identifying the genes that encode proteins involved in flavonoid biosynthesis in plants. The genes that code for these enzymes may be used to study the interactions among individuals of the pathways and develop methods to modulate the anthocyanin and flavonol biosynthetic pathways to control flavonoid biosynthesis. Accordingly, the availability of nucleic acid sequences encoding all or a portion of a FLS enzyme would facilitate studies to better understand flavonol biosynthesis in plants and provide genetic tools to enhance or otherwise alter flavonol and anthocyanin biosynthesis.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 80 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn flavonol synthase polypeptide of SEQ ID NO:2, a Momordica flavonol synthase polypeptide of SEQ ID NO:4, a Lamnanthes flavonol synthase polypeptide of SEQ ID NO:6, a soybean flavonol synthase polypeptide of SEQ ID NO:8, a wheat flavonol synthase polypeptide of SEQ ID NO:10. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7 and 9 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8 and 10. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7 and 9 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a flavonol synthase polypeptide of at least 80 amino acids comprising at least 85% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8 and 10.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a flavonol synthase polypeptide in a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a plant cell;
measuring the level a flavonol synthase polypeptide in the plant cell containing the isolated polynucleotide; and
comparing the level of a flavonol synthase polypeptide in the plant cell containing the isolated polynucleotide with the level of a flavonol synthase polypeptide in a plant cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a flavonol synthase polypeptide gene, preferably a plant flavonol synthase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7 and 9 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a flavonol synthase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a flavonol synthase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”).
Allen Stephen M.
Fader Gary M.
E. I. du Pont de Nemours & Company
Sisson Bradley L.
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