Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
1999-07-02
2001-08-14
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C536S023200, C435S252300, C435S006120, C435S320100, C435S190000, C435S468000, C800S278000, C800S295000
Reexamination Certificate
active
06274379
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 sorbitol biosynthetic enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
Sorbitol (D-glucitol) is an acyclic polyol found in a number of plant species (Kuo et al. (1990)
Plant Physiol
93:1514-1520). Sorbitol is the primary photosynthetic product in rosaceous fruits and can account for a major portion of the carbon transported from the leaf. In corn sorbitol is found in seed and silk but not in pollen and leaf and low amounts of sorbitol are detectable in developing corn kernels. Sorbitol is found in soybeans and it is suggested that the accumulation of sorbitol may play a role in facilitating hexose metabolism in germinating seedlings (Kuo et al. (1990)
Plant Physiol
93:1514-1520). During germination, soybeans convert oil and soluble oligosaccharides into sucrose which is in turn converted to glucose and fructose to fuel rapid growth. Some investigators have speculated that since plant fructokinases exhibit strong substrate inhibition by fructose, the presence of a sorbitol pathway may provide a mechanism to bypass this inhibition by converting excess fructose into sorbitol. This would help facilitate the metabolism of free glucose and fructose.
The metabolism of sorbitol has been extensively studied in several plant species (Kuo et al. (1990)
Plant Physiol
93:1514-1520, Loescher (1987)
Physiol. Plantarum
70:553-557, Loescher et al. in: Photoassimilate Distribution in Plants and Crops, ed. Zamski et al. Marcel Dekker, Inc., New York). There are several enzyme activities involved in sorbitol metabolism. Three of these enzymes are aldehyde reductase (NADPH-dependent aldose 6-phosphate reductase), sorbitol dehydrogenase, and NADP-dependent D-sorbitol-6-phosphate dehydrogenase. Aldehyde reductase appears responsible for the conversion of glucose to sorbitol; NADP-dependent D-sorbitol-6-phosphate dehydrogenase is also involved in sorbitol synthesis, and sorbitol dehydrogenase is involved in the conversion of sorbitol to fructose. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand carbohydrate metabolism and function in plants, provide genetic tools for the manipulation of the sorbitol biosynthetic pathway, and provide a means to control carbon partitioning in plant cells.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding sorbitol biosynthetic enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a sorbitol biosynthetic enzyme selected from the group consisting of aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase.
In another embodiment, the instant invention relates to a chimeric gene encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase.
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. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
TABLE 1
Sorbitol Biosynthetic Enzymes
SEQ ID NO:
(Amino
Protein
Clone Designation
(Nucleotide)
Acid)
Aldehyde reductase
Contig composed of:
1
2
ccase-b.pk0023.g2
ceb5.pk0015.g5
ceb5.pk0032.e9
ceb5.pk0044.c8
ceb5.pk0049.d10
ceb5.pk0052.a4
ceb5.pk0052.h8
ceb5.pk0062.a4
ceb5.pk0075.e7
ceb5.pk0075.g12
cen6.pk0001.g2
Aldehyde reductase
rca1n.pk022.j17
3
4
Aldehyde reductase
sr1.pk0003.c5
5
6
Aldehyde reductase
wl1n.pk0078.e5
7
8
NADP-dependent
p0002.cgevj66r
9
10
D-sorbitol-6-phosphate
dehydrogenase
NADP-dependent
rls2.pk0004.b8
11
12
D-sorbitol-6-phosphate
dehydrogenase
NADP-dependent
ses2w.pk0038.e12
13
14
D-sorbitol-6-phosphate
dehydrogenase
Sorbitol dehydrogenase
p0113.cieae77r
15
16
Sorbitol dehydrogenase
rlr6.pk0096.b8
17
18
Sorbitol dehydrogenase
sgs6c.pk001.122
19
20
Sorbitol dehydrogenase
wlm96.pk0016.h12
21
22
The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in
Nucleic Acids Research
13:3021-3030 (1985) and in the
Biochemical Journal
219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
DETAILED DESCRIPTION OF THE INVENTION
In the context of this disclosure, a number of terms shall be utilized. As used herein, a “nucleic acid fragment” is a polymer of RNA or DNA that
Famodu Layo O.
Kinney Anthony J.
Orozco Emil M.
E. I. du Pont de Nemours & Company
Monshipouri M.
Prouty Rebecca E.
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