Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-02-09
2003-04-22
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S006120, C435S069100, C435S183000, C435S410000, C435S419000, C435S320100, C530S350000, C530S370000, C536S023100, C536S023200, C536S023600, C536S024100, C536S024300, C536S024330, C800S295000
Reexamination Certificate
active
06552249
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 lignin biosynthetic enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
Plant cells and tissues can respond to mechanical, chemical or pathogen induced injury by producing various phenolic compounds including mono- or dimethoxylated lignin precursors derived from cinnamic acid via a complex series of biochemical reactions. These lignin precursors are eventually used by the plant to produce the phenolic macromolecule lignin which helps in wound repair by adding hydrophobicity, a physical barrier against pathogen infection and mechanical strength to the injured tissue (Vance, C. P., et al., 1980
, Annu Rev Phytopathol
18:259-288).
Cinnamyl-alcohol dehydrogenase (CAD) catalyzes the final step in the production of lignin monomers and has been implicated in plant defense response to fungal infection. Because of lignins importance in cell wall architecture and wound repair mechanisms there is considerable interest in the prospects for altering lignin quantity or quality by genetic engineering. For example, chemical treatments needed to remove lignin during the paper-pulping process are expensive and environmentally unfriendly. Plants with altered lignin quantity or quality could benefit this industry (Boudet, A., et al., 1996
, Mol Breeding
2:25-39; Campbell, M., et al., 1996
, Plant Physiol
110:3-13). Also, during soybean seed storage and processing lignin monomers are often oxidized, via polyphenol oxidase, to compounds that impart an undesirable yellow color to soybean seeds and seed products. It may be possible to reduce the amount of these phenolic compounds by suppressing CAD activity.
Cinnamyl-alcohol dehydrogenase genes described herein appear to be part of a multigene family. This application details the discovery of several different CAD genes from rice, soybean and wheat. Based on homology each of the genes (which range from 24% to 82% in similarity) have been placed into one of five different sub-groups that make up the CAD multigene family. There is a great deal of interest in identifying the genes that encode proteins involved in the production of lignin in plants. These genes may be used in plant cells to control lignin production. Accordingly, the availability of nucleic acid sequences encoding all or a portion of an enzyme involved in the production of lignin would facilitate studies to better understand lignin production in plants and provide genetic tools to enhance or otherwise alter lignin biosynthesis which in turn could provide mechanisms to control cell wall architecture, host defense and injury repair mechanisms and reduce the pool of phenolic 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 200 amino acids that has at least 92% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn cinnamyl-alcohol dehydrogenase polypeptide of SEQ ID NO:8, rice cinnamyl-alcohol dehydrogenase polypeptides of SEQ ID NOs:2 and 10, soybean cinnamyl-alcohol dehydrogenase polypeptides of SEQ ID NOs:4, 12, 20, 28, 30 and 34, and wheat cinnamyl-alcohol dehydrogenase polypeptides of SEQ ID NOs:6, 14 and 32. 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 103 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 cinnamyl-alcohol dehydrogenase polypeptide of SEQ ID NO:26, a rice cinnamyl-alcohol dehydrogenase polypeptide of SEQ ID NO:18 and wheat cinnamyl-alcohol dehydrogenase polypeptides of SEQ ID NOs:22 and 24. 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 186 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a corn cinnamyl-alcohol dehydrogenase polypeptide of SEQ ID NO:16. 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, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47 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, 46 and 48. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences 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:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47and 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 cinnamyl-alcohol dehydrogenase polypeptide of at least 200 amino acids comprising at least 92% 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, 10, 12, 14, 20, 28, 30, 32 and 34.
The present invention relates to a cinnamyl-alcohol dehydrogenase polypeptide of at least 103 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:18, 22, 24 and 26.
The present invention relates to a cinnamyl-alcohol dehydrogenase polypeptide of at least 186 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:16.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a cinnamyl-alcohol dehydrogenase 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 cinnamyl-alcohol dehydrogenase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a cinnamnyl-alcohol dehydrogenase polypeptide in the host cell containing the isolated polynucleotide with the level of a cinnamyl-alcohol dehydrogenase polypeptide in the host cell that does not contain the isolated polynucleotide.
The present inven
Cahoon Rebecca E.
Fader Gary M.
Rafalski J. Antoni
Bui Phuong T.
E.I. du Pont de Nemours and Company
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