Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-10-03
2004-12-14
Nelson, Amy J. (Department: 1638)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023200, C435S320100, C435S419000, C435S468000
Reexamination Certificate
active
06831168
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an aldehyde oxidase gene derived from a plant and utilization thereof.
It has been known that a natural plant growth hormone auxin alternatively IAA or indoleacetic acid is produced from tryptophane via indoleacetaldehyde followed by the action of anoxidase in higher plants. The hormone is deeply involved in various morphogenesis and environmental adaptation of a plant by its physiological activity and has significant effects on maturing by growth acceleration in general crop cultivation, improvement in yield and in quality by rooting acceleration in nursery plant production, increase in yield by growth acceleration of fruits in fruit vegetable cultivation, increase in added value by acceleration of flowering and elongation of life by prevention of defoliation or aging in ornamental plant cultivation. Therefore, there has been a strong demand for a method for artificially controlling the said enzyme for industry and particularly agricultural production.
Under these circumstances, the present inventors have successfully determined the total amino acid sequence and gene of the enzyme and completed the present invention.
Thus, the present invention provides:
1) An aldehyde oxidase gene which is a 4.4 Kbp gene obtainable from a plant and which encodes an amino acid sequence of an enzyme capable of oxidizing an aldehyde compound to a carboxylic acid (hereinafter, referred to as the gene of the present invention),
2) The aldehyde oxidase gene according to item 1), wherein the aldehyde compound is indoleacetaldehyde and the carboxylic acid is indoleacetic acid,
3) The aldehyde oxidase gene according to item 1 or 2 which is derived from a maize plant (
Zea mays L
.),
4) The aldehyde oxidase gene according to item 1 which is a nucleotide sequence encoding an amino acid sequence shown by SEQ ID NO: 2,
5) The aldehyde oxidase gene according to item 4 which has a nucleotide sequence shown by SEQ ID NO: 1 (loci of CDS being 46 . . . 4120),
6) The aldehyde oxidase gene according to item 1 which is a nucleotide sequence encoding an amino acid sequence shown by SEQ ID NO: 4,
7) The aldehyde oxidase gene according to item 6 which has a nucleotide sequence shown by SEQ ID NO: 3 (loci of CDS being 91 . . . 4138),
8) A plasmid comprising the aldehyde oxidase gene according to item 1, 2, 3, 4, 5, 6 or 7,
9) A transformant transformed by introducing the plasmid according to item 8 into a host cell,
10) The transformant according to item 9, wherein the host cell is a microorganism,
11) The transformant according to item 9, wherein the host cell is a plant,
12) A process for constructing an expression plasmid which comprises ligating:
(1) a promoter capable of functioning in a plant cell,
(2) an aldehyde oxidase gene according to item 1, 2, 3, 4, 5, 6 or 7 and
(3) a terminator capable of functioning in a plant in a functional manner and in the said order described above,
13) An expression plasmid comprising:
(1) a promoter capable of functioning in a plant cell,
(2) an aldehyde oxidase gene according to item 1, 2, 3, 4, 5, 6 or 7 and
(3) a terminator capable of functioning in a plant which are ligated in a functional manner and in the said order described above,
14) A process for controlling production of an aldehyde oxidase in a transformant which comprises introducing, into a host cell, an expression plasmid comprising:
(1) a promoter capable of functioning in a plant cell,
(2) an aldehyde oxidase gene and
(3) a terminator capable of functioning in a plant which are ligated in a functional manner and in the said order described above to transform said host cell,
15) The process according to item 14, wherein the aldehyde oxidase gene is derived from a plant and the host cell is a plant, and
16) The process according to item 14, wherein the expression plasmid is the expression plasmid according to item 13.
EMBODIMENTS OF THE INVENTION
The present invention will be described in more detail.
The gene of the present invention comprises about 4.4 kbp nucleotide obtainable from a plant and is an aldehyde oxidase gene that encodes an amino acid sequence of an enzyme capable of oxidizing an aldehyde compound to generate a carboxylic acid. For example, it is capable of oxidizing indoleacetaldehyde to generate indoleacetic acid.
The gene of the present invention can be obtained from a plant, for example, maize or the like. The gene of the present invention and the enzyme as the translation product of it have an action of oxidizing an acetaldehyde compound to a carboxylic acid in a cell. Said enzyme may also act, for example, on benzaldehyde, butyraldehyde, protocatechualdehyde or the like as the substrate, in addition to indolealdehyde of course, a single enzyme may act on plural compounds as substrates.
The gene of the present invention specifically includes, for example, a gene which is a nucleotide sequence encoding an amino acid sequence shown by SEQ ID NO: 2 and a gene which is a nucleotide equence encoding an amino acid sequence shown by SEQ ID NO: 4 as well as an equivalent of them. The expression “an equivalent of them” used herein means an aldehyde oxidase gene having a nucleotide sequence of an aldehyde oxidase gene that encodes an amino acid sequence shown by SEQ ID NO: 2 kor SEQ ID NO: 4 with a single nucleotide or plural nucleotides added, deleted or replaced, and refers to a DNA which is an analog having the same function. More particularly, this includes a gene having a nucleotide sequence shown by SEQ ID NO: 1 (loci of CDS being 46 . . . 4120) or a nucleotide sequence shown by SEQ ID NO: 3 (loci of CDS being 91 . . . 4138).
The gene of the present invention can be obtained by the following process.
For example, seeds of Golden Cross Bantam 70 (purchased from Sakata-no-tane), a maize cultivar, are subjected to a treatment for hastening of germination by immersing overnight in running tap water, subsequently seeded on a paper towel moistened with water and placed in red light (0.8 W/m
2
) under a condition of 25° C. for 2 days and then in the dark for 1 day to allow germination. Top portions of young sheaths grown to 1.0-1.5 cm from the obtained seedlings are excised under a green safety light, immediately frozen with liquid nitrogen and stored at −30° C. as samples for purification of enzymes and samples for extracting RNAs.
For purifying aldehyde oxidase from the frozen samples prepared in this manner, it is appropriate to use a method described in T. Koshiba et al., Plant Physiology, 1996, 110, 781-789.
In order to prevent decrease in activity of the enzyme and decomposition of the protein during procedures of extraction and purification, it is preferred to carry out all the treatments in the purification steps at a lower temperature of 2-4° C., as is ordinary manner in such procedures. First, 150-200 g of the frozen sample is taken as a material for one batch of purification. The material is mechanically crushed by a homogenizer or the like with addition of 400 ml of 0.1 M phosphate buffer (pH 7.4) and centrifuged at 12,000 g for 30 minutes. The supernatant is separated as a crude enzyme standard sample. From the crude enzyme standard sample, a fraction is obtained with 30-50% saturated ammonium sulfate, dialyzed against 20 mM Tris HCl buffer (pH 8.0) and centrifuged at 20,000 g for 20 minutes. The supernatant from centrifugation is passed over an ion-exchange column (for example, DEAE TOYOPEARL 650 M, manufactured by Tosoh) and a fraction with an aldehyde oxidase activity is collected. Said fraction with the specific activity is subjected to chromatography with a hydrophobic column, a hydroxyapatite column and an ion-exchange column (for example, DEAE-5PM) in this order and purified until the fraction with aldehyde oxidase activity is detected as an almost single protein band by silver staining after electrophoresis.
According to the above described purification procedure, about 2,000 times purification, in terms of the amount of protein in the crude enzyme standard sample, is usually possible. It can be confirmed that the final
Birch & Stewart Kolasch & Birch, LLP
Kallis Russell
Nelson Amy J.
Sumitomo Chemical Company Limited
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