Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2001-07-16
2004-01-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S183000, C435S252300, C435S320100, C435S071100, C536S023200, C536S023100
Reexamination Certificate
active
06680185
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 polyphenol oxidase enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
Polyphenol oxidase (PPO) catalyzes the oxidation of mono- and O-diphenols to O-diquinones. The oxidation of mono- and diphenols, which occurs during fruit ripening and plant wounding, produces and undesirable browning of fruit and vegetable material (Hunt M. D., et al. 1993
, Plant Mol. Biol
. 21(1):59-68). Inhibition of polyphenol oxidase activity would likely prevent the accumulation of the brown discoloration in fruits and may improve flavor. Furthermore, polyphenols function as antioxidants; inhibition of polyphenol oxidase would increase the level of polyphenols in fruits and vegetables and thus add food value.
In plants polyphenol oxidase activity appears to be encoded by a multigene family. For example, in tomato seven nuclear genes have been reported that encode PPO activity (Newman S. M., et al., 1993
, Plant Mol. Biol
. 21(6):1035-1051). The nucleic acid fragments described herein also appear to encode several different PPO enzymes. Based on amino acid homology, seven different PPO types (that share less than 75% similarity at the amino acid level) have been identified in soybean cDNA libraries. Nucleic acid fragments encoding a two types of PPO enzymes from corn and one type of PPO from wheat are also described. The various types of PPO enzymes have been designated A-I.
There is a great deal of interest in identifying the genes that encode proteins involved in polyphenol oxidation in plants. These genes may be used in plant cells to control the oxidation of phenolic compounds that impart discoloration to fruit and vegetables. The genes may also be used to increase the level of antioxidants in fruits and vegetables. Accordingly, the availability of nucleic acid sequences encoding all or a portion of a PPO enzyme would facilitate studies to better understand polyphenol oxidation in plants and provide genetic tools to inhibit or otherwise alter PPO activity which in turn could provide mechanisms to control discoloration in fruits and vegetables and increase the pool of antioxidant compounds in plant cells.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 112 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of corn polyphenol oxidase polypeptides of SEQ ID NOs:4, 38 and 42, soybean polyphenol oxidase polypeptides of SEQ ID NOs:6, 24 28, 32, 34, 36 and 44, and a wheat polyphenol oxidase polypeptide of SEQ ID NO: 14. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 163 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn polyphenol oxidase polypeptide of SEQ ID NO:40, and soybean polyphenol oxidase polypeptides of SEQ ID NOs:10 and 20. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 8, 12, 16, 18, 22, 30 and 46.
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, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46. 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, most preferably at least one of 15) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:3, 5, 9, 13, 19, 23, 27, 31, 33, 35, 37, 39, 41, and 43 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 polyphenol oxidase polypeptide of at least 112 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 14, 24, 32, 34, 36, 38, 42 and 44.
The present invention relates to a polyphenol oxidase polypeptide of at least 163 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:10, 20 and 40.
The present invention also relates to a polypeptide of at least 50 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 8, 12, 16, 18, 22, 30 and 46.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a polyphenol oxidase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level a polyphenol oxidase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a polyphenol oxidase polypeptide in the host cell containing the isolated polynucleotide with the level of a polyphenol oxidase polypeptide in the host 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 polyphenol oxidase polypeptide gene, preferably a plant polyphenol oxidase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:3, 5, 9, 13, 19, 23, 27, 31, 33, 35, 37, 39, 41, and 43 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 polyphenol oxidase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fra
Cahoon Rebecca E.
Falco Saverio Carl
Kinney Anthony J.
Miao Guo-Hua
Achutamurthy Ponnathapu
E. I. du Pont de Nemours and Company
Pak Yong
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