Invertase inhibitor

Details Invertase inhibitor Invertase inhibitor

1999-04-05

2002-05-07

Nelson, Amy J. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters carbohydrate production in the plant

C800S284000, C800S278000, C800S294000, C800S295000, C800S298000, C800S317000, C800S317200, C800S317400, C435S069100, C435S069200, C435S419000, C435S468000, C435S320100, C435S069700, C435S252300, C435S430000, C536S021000, C536S023600, C536S024100

Reexamination Certificate

active

06384300

ABSTRACT:

This invention relates to a nucleic acid that contains at least one nucleic acid sequence coding for a polypeptide, said polypeptide being capable of reducing the enzymatic activity of an invertase, the polypeptide itself, as well as transgenic plants that contain this nucleic acid sequence. The invention further relates to methods of preparing such transgenic plants with reduced storage sucrose loss.
BACKGROUND OF THE INVENTION
During the storage of sugar beets (
Beta vulgaris
), in the period between harvest and processing, respiration or sucrose metabolism leads to a sucrose loss of roughly 0.02% per day. This loss is further accompanied by a significant diminution of quality as a consequence of the increase of reducing sugars, in particular fructose and glucose (Burba, M. (1976), “Respiration and Sucrose Metabolism of Sugar Beets in Storage,”
Zeitschrift für die Zuckerindustrie
26:647-658). The first metabolic step in the breakdown of sucrose during the storage of beets is enzymatic hydrolysis by a vacuolar invertase. This enzyme is synthesized de novo in the beet tissue after injury (Milling, R. J., Leigh, R. A., and Hall, J. L. (1993), “Synthesis of a Vacuolar Acid Invertase in Washed Discs of Storage Root Tissue of Red Beet (
Beta vulgaris
L.),
J. Exp. Bot.
44:1687-1694). Because the bulk of beet sucrose is localized in the vacuoles of the cell, the (injury-)induced vacuolar invertase plays a central role in storage sucrose loss.
At present there is no satisfactory solution to the problem of storage sucrose losses (Burba, 1976). The most important practices in the prior art consist in maintaining low temperatures (below 12° C.) and a well-defined atmospheric humidity (between 90 and 96%). All practices used up to now to reduce the storage losses are, however, unsatisfactory.
Conversion of sucrose to the hexoses glucose and fructose in storage, and thus loss of sucrose, also occurs during the “cold sweetening” of potatoes. As a result of cold processing, a vacuolar invertase is induced in the potato tubers and determines the ratio of sucrose to hexoses (Zrenner, R., Schüler, K., and Sonnewald, U. (1996), “Soluble Acid Invertase Determines the Hexose-to-Sucrose Ratio in Cold-Stored Potato Tubers,”
Planta
198:246-252). The formation of hexoses as a result of cold sweetening leads to diminutions of quality in the making of, for example, French-fried potatoes.
Tomato fruits (
Lycopersicon esculentum
Mill.) exhibit a high water content. This is due in part to the osmotically active endogenous sugars (sucrose and hexoses). Lowering the total sugar content by means of inhibiting the invertase-mediated hydrolysis of sucrose leads to smaller fruits with lower water content (Klann, E. M., Hall, B., and Bennett, A. B. (1996), “Antisense Acid Invertase (TIV1) Gene Alters Soluble Sugar Composition and Size in Transgenic Tomato Fruit,”
Plant Physiology
112:1321-1330). Reducing the water content of the tomato fruits leads to a saving in energy costs for the production of fruit concentrates (e.g., ketchup). Because the reduction of vacuolar invertase activity via invertase antisense expression is incomplete because of the occurrence of a variety of isoforms, the transgenic introduction of an invertase inhibitor might result in great advantages, in particular if said invertase inhibitor has an equal inhibiting action on these various isoforms.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to create a new system that causes essentially no sucrose storage-related losses in plants.
This object is achieved by virtue of the subject matters of the invention characterized in the Claims.
DETAILED DESCRIPTION
A first subject matter of the invention relates to a nucleic acid that contains at least one nucleic acid sequence coding for a polypeptide, which polypeptide is capable of reducing or lowering the enzymatic activity of an invertase.
The terms “nucleic acid” and “nucleic acid sequence” denote natural or semisynthetic or synthetic or modified nucleic acid molecules from deoxyribonucleotides and/or ribonucleotides and/or modified nucleotides.
The term “polypeptide” denotes naturally occurring polypeptides and recombinant polypeptides. Recombinant polypeptides denote a construct prepared by molecular-biological techniques, based on the natural DNA of the original genome or the natural DNA modified with a foreign DNA sequence, which construct can be recombined, for example with plasmids, and replicated and expressed in a suitable host system.
The expression “a polypeptide capable of reducing the enzymatic activity of an invertase” denotes a polypeptide that, in the process of binding to an invertase, reduces the enzymatic activity of said invertase, complete inhibition being possible if there is a sufficient quantity of the inhibitor protein. A roughly 90% inhibition of the vacuolar invertase is preferably to be achieved by means of the inhibitor expression in the transgenic plant.
In a embodiment of the invention, the invertase in a plant cell is vacuolarly localized. In another embodiment, the invertase is localized in the cell wall. In a further embodiment, the invertase is localized in the cytosol. The invertase is preferably derived from sugar beet, potato or tomato.
In a preferred embodiment of the invention, the nucleic acid comprises the nucleic acid sequences shown in FIGS.
1
(
a
)-
1
(
d
) (SEQ ID No. 1),
3
(SEQ ID No. 2),
12
(SEQ ID No. 3) and
14
(
a
)-(
b
) (SEQ ID No. 4) or segments or fragments thereof as well as nucleic acid sequences that can hybridize with the complementary sequences of the nucleic acid sequences shown in FIGS.
1
(
a
-(
d
),
3
,
12
or
14
(
a
)-(
b
) or segments or fragments thereof.
In another embodiment, the nucleic acid according to the invention contains a further nucleic acid sequence coding for a targeting sequence. The term “targeting sequence” denotes an amino acid sequence that mediates cellular targeting into a well-defined cellular compartment, for example targeting into the vacuoles.
In a preferred embodiment of the invention, the targeting sequence comprises the vacuolar targeting sequence of barley lectin having the following amino acid sequence:
SEQ ID NO: 9 LEGVFAEIAASNSTLVAE
In another embodiment, the nucleic acid according to the invention contains a further nucleic acid sequence coding for a signal peptide. The term “signal peptide” denotes a hydrophobic amino acid sequence that is recognized by the signal recognition particle (SRP). The SRP mediates the synthesis of the entire polypeptide on the rough endoplasmic reticulum (ER), with the consequence that the resulting polypeptide is released into the ER lumen.
In a further embodiment, the nucleic acid according to the invention contains a nucleic acid sequence coding for an ER retention sequence.
In a preferred embodiment, the signal peptide is derived from an invertase, preferably from cell-wall invertase from tobacco.
In another embodiment of the invention, the nucleic acid contains a further nucleic acid sequence that comprises a promoter suitable for expression in plants. This promoter or promoter sequence is preferably derived from the same plant as the invertase. In an especially preferred embodiment of the invention, the promoter is a promoter specific to potato or sugar beet.
In summary, the nucleic acid according to the invention can comprise the above-defined nucleic acid sequence coding the polypeptide and, if appropriate, the above-defined nucleic acid sequence coding a targeting sequence and/or the above-defined promoter, where all nucleic acid sequences coding an amino acid sequence are preferably arranged in the reading frame and can be degenerated in accordance with the genetic code.
A further subject matter of the invention is a vector that contains the above-defined nucleic acid according to the invention for the expression of the recombinant polypeptide in prokaryotic or eukaryotic host cells. The vector according to the invention can preferably contain suitable regulatory elements such as promoters, enhancers, termination sequence

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