Grain processing method and transgenic plants useful therein

Details Grain processing method and transgenic plants useful therein Grain processing method and transgenic plants useful therein

2000-06-21

2003-03-11

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S287000, C800S312000, C800S320100, C536S023600, C435S069100, C435S189000, C435S320100, C435S412000, C435S415000, C435S419000

Reexamination Certificate

active

06531648

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to improved methods of grain processing to enhance protein and starch recovery, particularly in corn wet milling and soybean processing, as well as novel transgenic plants useful in such processes.
BACKGROUND OF THE INVENTION
Thioredoxin (TRX) and thioredoxin reductase (TR) are enzymes that use NADPH to reduce disulphide bonds in proteins. Protein disulphide bonds play an important role in grain processing efficiencies and in the quality of the products recovered from grain processing. Development of effective ways to eliminate or decrease the extent of protein disulphide bonding in grain would increase processing efficiencies. Additionally, grain and grain-derived product performance in livestock feed are also affected by inter- and intramolecular disulphide bonding. Grain digestibility, nutrient availability and the neutralization of anti-nutritive factors (e.g., protease, amylase inhibitors etc.) would be increased by reducing the extent of disulphide bonding. See, PCT/EP99/09986, filed Dec. 15, 1999, and U.S. Provisional Application No. 60/183,051, filed Dec. 17, 1998, both of which are incorporated herein by reference.
SUMMARY OF THE INVENTION
Expression of transgenic thioredoxin and/or thioredoxin reductase in corn and soybeans and the use of thioredoxin in grain processing, e.g., wet milling, is novel and provides an alternative method for reducing the disulfide bonds in seed proteins during or prior to industrial processing. The invention therefore provides grains with altered storage protein quality as well as grains that perform qualitatively differently from normal grain during industrial processing or animal digestion (both referred to subsequently as “processing”).
This method of delivery of thioredoxin and/or thioredoxin reductase eliminates the need to develop exogenous sources of thioredoxin and/or thioredoxin reductase for addition during processing. A second advantage to supplying thioredoxin and/or thioredoxin reductase via the grains is that physical disruption of seed integrity is not necessary to bring the enzyme in contact with the storage or matrix proteins of the seed prior to processing or as an extra processing step.
Three modes of thioredoxin utilization in grain processing are provided:
1. Expression and action during seed development to alter the composition and quality of harvested grain;
2. Expression (but no activity) during seed development to alter the quality of the products upon processing;
3. Production of thioredoxin and/or thioredoxin reductase in grain that is used to alter the quality of other grain products by addition during processing.
The invention described herein is applicable to all grain crops, in particular corn, soybean, wheat, and barley, most particularly corn and soybean, especially corn. Expression of transgenic thioredoxin and/or thioredoxin reductase in grain is a means of altering the quality of the material (seeds) going into grain processing, altering the quality of the material derived from grain processing, maximizing yields of specific seed components during processing (increasing efficiency), changing processing methods, and creating new uses for seed-derived fractions or components from milling streams.
The invention thus provides a plant which expresses a thioredoxin and/or thioredoxin reductase, e.g. a thioredoxin and/or thioredoxin reductase not naturally expressed in plants, for example a plant comprising a heterologous DNA sequence coding for a thioredoxin stably integrated into its nuclear or plastid DNA, preferably under control of an inducible promoter, e.g., a chemically-inducible promoter, for example either operatively linked to the inducible promoter or under control of transactivator-regulated promoter wherein the corresponding transactivator is under control of the inducible promoter or is expressed in a second plant such that the promoter is activated by hybridization with the second plant; wherein the thioredoxin or thioredoxin reductase is preferably thermostable; such plant also including seed therefor, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use, and seed harvested therefrom, e.g., for use in a milling process as described above.
The transgenic plant of the invention may optionally further comprise genes for enhanced production of thioredoxin reductase and/or NADPH.
The invention further provides a method for producing a thioredoxin comprising cultivating a thioredoxin-expressing plant as described above; a method for producing starch and/or protein comprising extracting starch or protein from seed harvested from a plant as described above; and a method for wet milling comprising steeping seed from a thioredoxin-expressing plant as described above and extracting starch and/or protein therefrom.
The invention further provides a plant expressible expression cassette comprising a coding region for a thioredoxin or thioredoxin reductase, preferably a thioredoxin derived from a thermophilic organism, e.g., from an archea, for example from
Methanococcus jannaschii
or
Archaeglobus fulgidus
, e.g., as described below, wherein the coding region is preferably optimized to contain plant preferred codons, said coding region being operatively linked to promoter and terminator sequences which function in a plant, wherein the promotor is preferably a seed specific promoter or an inducible promoter, e.g., a chemically inducible or transactivator-regulated promoter; for example a plastid or nuclear expressible expression cassette comprising a promoter, e.g., a transactivator-mediated promoter regulated by a nuclear transactivator (e.g., the T7 promoter when the transactivator is T7 RNA polynerase the expression of which is optionally under control of an inducible promoter).
The invention further provides a vector comprising such a plant expressible expression cassette.
The invention further provides a plant transformed with such a vector, or a transgenic plant which comprises in its genome, e.g., its nuclear or plastid genome, such a plant expressible expression cassette.
The invention also comprises a method of producing grain comprising high levels of thioredoxin or thioredoxin reductase comprising pollinating a first plant comprising a heterologous expression cassette comprising a transactivator-mediated promoter regulated and operatively linked to a DNA sequence coding for a thioredoxin or thioredoxin reductase, the first plant preferably being emasculated or male sterile, with pollen from a second plant comprising a heterologous expression cassette comprising a promoter operatively linked to a DNA sequence coding for a transactivator capable of regulating said transactivator-mediated promoter, and recovering grain from the plant thus pollinated.
The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding an Arabidopsis NADPH
+
dependent thioredoxin reductase (NTR), wherein the nucleotide sequence is optimized for expression in a monocotyledonous plant, preferably optimized for expression in maize. The nucleotide sequence is preferably the nucleotide sequence of SEQ ID NO:24 and preferably encodes the amino acid sequence of SEQ ID NO:25.
The invention also provides an isolated nucleic molecule comprising a nucleotide sequence encoding a rice NADPH
+
dependent thioredoxin reductase (NTR). The nucleotide sequence preferably encodes the amino acid sequence of SEQ ID NO:27. The nucleotide sequence is preferably the nucleotide sequence of SEQ ID NO:25.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO:1—Protein sequence of thioredoxin from
Methanococcus jannaschii
(gi|1591029).
SEQ ID NO:2—Protein sequence of thioredoxin from
Archaeoglobus fulgidus
(gi|2649903)(trx-1).
SEQ ID NO:3—Protein sequence of thioredoxin from
Archaeoglobus fulgidus
(gi|2649838) (trx-2).
SEQ ID NO:4—Protein sequence of thioredoxin from
Archaeoglobus fulgidus
(gi|2649295) (trx-3).
SEQ ID NO:5—Protein sequence of thioredoxin from

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