MXPA01006050A - Thioredoxin and grain processing - Google Patents

Abstract

The invention provides methods of processing grain, particularly corn and soybeans, utilizing thioredoxin and/or thioredoxin reductase to enhance extractability and recovery of starch and protein. The invention further provides transgenic plants expressing thermostable thioredoxin and/or thioredoxin reductase.

Description

TIORREDOXIN AND GRAIN PROCESSING Description of the Invention This invention relates to novel methods for the processing of grains, in order to improve the recovery of protein and starch, particularly in the wet milling of corn, and in the processing of soybeans. , as well as novel transgenic plants useful in these processes. Thioredoxin (TRX) and thioredoxin reductase (TR) are enzymes that use NADPH to reduce disulfide bonds in proteins. Protein disulfide bonds play an important role in the efficiencies of grain processing, and in the quality of the products recovered from grain processing. The development of effective ways to eliminate or decrease the degree of protein disulfide bond in the grains will increase the processing efficiencies. Additionally, grain performance in livestock feed is also affected by the inter- and intra-molecular disulfide bond: grain digestibility, nutrient availability, and neutralization of anti-nutrient factors (eg, protease) would be increased. , amylase inhibitors, etc.), reducing the degree of connection of REF .: 129931 disulfide. The expression of transgenic thioredoxin and / or thioredoxin reductase in maize and soybeans, and the use of thioredoxin in the processing of grains, for example, wet milling, is novel and provides an alternative method to reduce protein binding. disulfide in seed proteins during industrial processing. Accordingly, the invention provides grains with an altered storage protein quality, as well as grains that perform in a qualitatively different manner from the normal grain during industrial processing or animal digestion (both subsequently referred to as "processing"). This thioredoxin and / or thioredoxin reductase delivery method eliminates the need to develop exogenous sources of thioredoxin and / or thioredoxin reductase, to be added during processing. A second advantage of supplying thioredoxin and / or thioredoxin reductase by means of the grains, is that the physical alteration of the integrity of the seeds is not necessary to put the enzyme in contact with the storage or matrix proteins of the seeds before of processing, or as an extra step to processing.

Three modes of use of thioredoxin are provided in the processing of grains: 1) Expression and action during the development of the seeds to alter the composition and quality of the harvested grain; 2) Expression (but no activity) during the development of the seeds, to alter the quality of the products after processing; 3) Production of thioredoxin and / or thioredoxin reductase in the grain, which is used to alter the quality of other grain products by addition during processing. Therefore, the present invention provides: A method for increasing the separation efficiency of starch and protein in a grain milling process, which comprises impregnating the grain at an elevated temperature, in the presence of thioredoxin and / or complementary thioredoxin reductase, and separate the starch and protein components from the grain. A method as mentioned hereinabove, wherein the grain includes grain from a transgenic plant, wherein the transgene expresses thioredoxin and / or thioredoxin reductase, particularly a thioredoxin and / or thermostable thioredoxin reductase. - A method as mentioned hereinabove, wherein the plant is selected from dicotyledonous or monocotyledonous plants, particularly from cereals, and still in a more particular way, from maize (Zea mays) and bean soy. The invention also provides transgenic plants. In particular, the invention provides: A plant comprising a heterologous DNA sequence encoding a thioredoxin and / or thioredoxin reductase stably integrated into its Nuclear or plastid DNA. - A plant as mentioned above, wherein thioredoxin and / or thioredoxin reductase is thermostable. - A plant as mentioned above, wherein thioredoxin and / or thioredoxin reductase is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. - A plant as mentioned hereinabove, where the plant is selected from corn and soybeans.

The invention also provides: An expression cassette expressible in plants, comprising a coding region for a thioredoxin and / or thioredoxin reductase, operably linked to the promoter and terminator sequences that function in a plant. - An expression cassette expressible in plants as mentioned above, wherein thioredoxin and / or thioredoxin reductase is thermostable. An expression cassette expressible in plants, wherein thioredoxin and / or thioredoxin reductase is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO , SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 The invention further provides: A method for producing beads comprising high levels of thioredoxin and / or thioredoxin reductase, which comprises transforming plants with a cassette of expression as mentioned above. - A method for producing grains comprising high levels of thioredoxin and / or thioredoxin reductase, which comprises: Pollining a first plant comprising a cassette of heterologous expression, comprising a promoter regulated by transactivator, regulated and operatively linked to a sequence of DNA encoding a thioredoxin and / or thioredoxin reductase, with pollen from a second plant comprising a heterologous expression cassette comprising a promoter operably linked to a DNA sequence encoding a transactivator capable of regulating the promoter regulated by transactivator; and recover the grain of the plant so pollinated. In addition, the invention provides: The use of plants or plant material according to the invention, as animal feed. The invention described herein is applicable to all grain crops, in particular corn, soybeans, wheat, and barley, more particularly corn and soybeans, especially corn. The expression of thioredoxin and / or reductase of transgenic thioredoxin in the grain, is a means to alter the quality of the material (seeds) that goes to the processing of grains, alter the quality of the material derived from the grain processing, maximize the yields of the specific seed components during processing (increase efficiency), change processing methods, and create new uses for fractions or seed-derived components from grinding currents. Wet milling. Wet milling is a process to separate the starch, the protein, and the oil components from the grains, most often cereals, for example maize. It differs in the present from dry grinding, which is simply pulverizing the grain. The first step in wet grinding is usually the impregnation where the grain is soaked in water under carefully controlled conditions to soften the seeds and facilitate the separation of the components. Embryos carrying oil float to the surface of the aqueous solution, and are removed, and by a process of soaking and dehydration, milling, sorting, centrifugation, and washing, the starch is separated from the protein, and purified. The key difficulty is loosening the starch granules of the complicated protein matrix and the cell wall material that forms the grain endosperm. It is believed that one reason for this difficulty is the presence of inter- or intramolecular disulfide bonds., which make the protein matrix less soluble and less susceptible to proteolytic enzymes, and inhibit the release of starch granules from the protein matrix in the grain. At present, the primary means to reduce these bonds is to impregnate the grain in the presence of sulfur dioxide, but this is expensive, not suitable for the environment, and not optimally effective. Certain mutations exert beneficial effects on the protein matrix of the endosperm of the corn seed (mealy and opaque), but damage the integrity of the seed. The expression of transgenic thioredoxin provides some of these advantages without creating some of the seed integrity problems associated with these mutations. Activity after harvest or dependent on the processing of thioredoxin, has equally beneficial effects. For example, in one embodiment, the thioredoxin enzymes are directed and accumulated in the cell compartments. The reduction of the protein occurs immediately after the physical alteration of the seeds. In another modality, the thioredoxin of the passive endosperm is activated upon impregnation. In a preferred embodiment, the invention provides a plant that expresses a thioredoxin and thioredoxin reductase of transgenic thermostable thioredoxins, for example a thioredoxin and thioredoxin reductase derived from a hyperthermophilic organism, such that thioredoxin and thioredoxin reductase are not significantly active, except at high temperatures (for example, greater than 50 ° C). In one embodiment, thioredoxin and thioredoxin reductase thermostable are synergistic saccharification by expressing other thermostable enzymes in the endosperm.

Applications in Food The expression of thioredoxin and / or reductase of transgenic thioredoxin in grains is also useful to improve the characteristics of the grains, associated with digestibility, particularly in animal feeds. The susceptibility of the proteins of the food to the proteases is a function of the time and the conformation of the protein. Seed disintegration is often used in food formulation, such as steam flake formation. Both processes are designed to help the digestibility of the seeds. Softer seeds are desirable whose integrity can be more easily altered in the stomachs of animals. The conformational limitations and the cross-links between the proteins are important determinants of protease susceptibility. The modification of these bonds through a greater expression of thioredoxin helps in this way to digestion. Dry / Mass Grinding The content and quality of the protein are important determinants in flake production and mass production. The reduction of the disulfide bonds alters the nature of the corn meal, such that it is suitable for use as a substitute for wheat, especially flours made from high-protein white maize varieties. Soybean Crushing More than half of the US soybean crop is crushed or ground, and the quality of the protein in the low-fat soybean meal or resulting defatted soybean meal (or semolina) is important for subsequent processing. The yield and quality of protein from soy bean processing streams are economically important, and depend largely on the conformation of the protein. The increase in thioredoxin activity through the expression of thioredoxin and / or transgenic thioredoxin reductase increases the solubility of the protein, and therefore increases the yield, in the water soluble protein fractions. Recovery is facilitated by aqueous extraction of the defatted soybean meal under basic conditions. The improvement of thioredoxin activity through the expression of thioredoxin and / or transgenic thioredoxin reductase, also reduces the pH required for efficient extraction, and thus, reduces calcium or sodium hydroxide inlets, as well as low acid input for subsequent acid precipitation, allowing efficient recovery of proteins without alkaline damage, and reducing water consumption and waste effluents from the processing plant (which contain substantial loads of biological oxygen demand) . The reduced-oxidation state of the protein affects important functional properties supplied by the soy proteins, such as solubility, water absorption, viscosity, cohesion / adhesion, gelation, and elasticity. The removal of fiber during the production of the soy protein concentrate and hydrolysis of the soy protein isolate by the proteases is improved by increasing the thioredoxin activity, as described herein. In a similar manner, as described for maize previously, the increase of thioredoxin activity through the expression of thioredoxin and / or transgenic thioredoxin reductase, improves the functionality of active soybean meal in enzymes, and digestibility of the fraction of soybean meal and of the fraction of flake formation by steam in animal feed. Modification of the quality of the protein is provided during the development of the seeds and during processing, although it is preferred that the thioredoxin and / or reductase of transgenic thioredoxin be directed towards a cell compartment, and be thermostable, as described above., to avoid significant adverse effects after storage, and protein accumulation will be found as a result of thioredoxin activity during the development of the seeds. Alternatively, thioredoxin can be added as a processing enzyme, because it is not necessary (in contrast to wet milling of corn) to break the disulfide bonds until after the grain integrity is destroyed (crushing and extraction of oil).

Selection of thioredoxin and thioredoxin reductase for heterologous expression: thioredoxin genes, thioredoxin reductase, and protein disulfide isomerase (PDI) are found in eukaryotes, eubacteria as well as archaea, including hyperthermophilic organisms, such as th anococcus j anna schi i and Arch a eogl obu s ful gi du s. The selection of a particular gene depends in part on the desired application. For the methods of the present invention, the preferred thioredoxins have the following characteristics. 1. Heat Stability It is found that thioredoxin and related proteins from hyperthermophiles, have greater stability at high temperatures (> 50 ° C), and a relatively low activity at ambient temperatures. The expression of TRX and / or TR from hyperthermophiles, for example from archaea, such as Methanococcus j annaschi i and Archa eogl obus ful gi dus, or other hyperthermophiles, is preferred for expression during the development of seeds, such so that thioredoxin activity is not markedly increased until the grain is impregnated or processed at an elevated temperature. Most grain processing methods involve, or are compatible with, a high temperature step. Accordingly, thioredoxin and thermostable thioredoxin reductase are preferred. Thermostable means that the enzyme is preferably active at high temperatures, for example, temperatures higher than 40 ° C, more preferably higher than 50 ° C, for example 45 ° C to 60 ° C for wet milling, or still higher , for example from 45 ° C to 95 ° C. 2. Substrate Specificity It is also possible to reduce the undesirable effects on seed development by selecting a thioredoxin that acts preferentially on certain proteins, such as the structural protein in the matrix, and that has a low activity with the essential metabolic enzymes. . It has been shown that different TRXs differ in their reactivity with enzymes that are under the control of reduction-oxidation. Accordingly, it is possible to select a TRX that primarily acts on desired objectives, minimizing the undesirable side effects of overexpression. Suitable thermostable thioredoxin thioredoxins and reductases include the following: Thioredoxin sequence from Me thanococc us jannaschii (SEQ ID NQ: 1; gi | 1591029) MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPQKAMEYGIMA VPTIVINGDVEFIGAPTKEALVEAIKKRL Thioredoxin sequence from Archaeoglobus fulgidus (SEQ ID NQ: 2; gi | 2649903) (trx-1) MPMVRKAAFYAIAVISGVLAAVVGNALYHNFNSDLGAQAKIYFFYSDSCPHCREVK PYVEEFAKTHNLTWCNVAEMDANCSKIAQEFGIKYVPTLVIMDEEAHVFVGSDEVR TAIEGMK Thioredoxin sequence from Archaeoglobus fulgidus (SEQ ID NO: 3; gi | 2649838) (trx-2) MVFTSKYCPYCRAFEKVVERLMGELNGTVEFEVVDVDEKRELAEKYEVLMLPTLVL ADGDEVLGGFMGFADYKTAREAILEQISAFLKPDYKN Thiorredoxin sequence from Archaeoglobus fulgidus (SEQ ID NOM; gi | 2649295) (trx-3) MDELELIRQKKLKEMMQKMSGEEKARKVLDSPVKLNSSNFDETLKNNENVVVDF A EWCMPCKMIAPVIEELAKEYAGKVVFGKLNTDENPTI ARYGISAIPTLIFFKKGK PVDQLVGAMPKSELKRWVQRNL Thioredoxin sequence from Archaeoglobus fulgidus (SEQ ID NO: 5; gi | 2648389) (trx-4) MERLNSERFREVIQSDKLVVVDFYAD CMPCRYISPILEKLSKEYNGEVEFYKLNV DENQDVAFEYG1AS1PTVLFFRNGKVVGGFIGAMPESAVRAEIEKALGA sequence reductase thioredoxin (trxB) from Methanococcus j annaschii (SEQ ID NO: 6: gi | 1592167) MIHDTIIIGAGPGGLTAGIYAMRGKLNALCIEKENAGGRIAEAGIVENYPGFEEIR GYELAEKFKNHAEKFKLPIIYDEVIKIETKERPFKVITKNSEYLTKTIVI TGTKP KKLGLNEDKFIGRGISYCTMCDAFFYLNKEVIVIGRDTPAIMS INLKDI KKVIV ITDKSELKAAESIMLDKLKEANNVEIIYNAKPLEIVGEERAEGVKISVNGKEEIIK ADGIFISLGHVPNTEFLKDSGIELDKKGFIKTDENCRTNIDGIYAVGDVRGGVMQV AKAVGDGCVAMANIIKYLQKL sequence reductase thioredoxin from Arch to eogl obu s ful gi du s (SEQ ID NO: 7: gi | 2649006) (trxB) MYDVAIIGGGPAGLTAALYSARYGLKTVFFETVDPVSQLSLAAKIENYPGFEGSGM ELLEKMKEQAVKAGAEWKLEKVERVERNGETFTVI EGGEYEAXAIIVATGGKHKE AGIEGESAFIGRGVSYCATCDGNFFRGKKVIVYGSGKEAIEDAIYLHDIGCEVTIV SRTPSFRAEKLVEEVEKRGIPVHYSTTIRKIIGSGKVEKVVAYNREKKEEFEIEAD GIFVAIGMRPATDVVAEI.GVERDSMGYIKVDKEQRTNVEGVFAAGDCCDNPLKQVV TACGDGVAAYSAYKYLTS The genes encoding these proteins for use in the present invention, preferably are designed by back translation using codons preferred by the plant, to improve the G-C content and remove deleterious sequences, as described more fully below. The activity of the proteins can be improved by mixing the DNA or other means, as described below. Accordingly, the invention comprises proteins derived from these proteins, especially proteins that are substantially similar, which retain the thioredoxin or thioredoxin reductase activity. For the design of the expression of thioredoxin in seeds for the activity during the development of the grain, the promoters that direct the specific expression of the TRX and TR seeds are preferred, because it is the storage direction, in such a way that the The enzyme will have the desired effects on the proteins in storage, which may be desirable in some applications. However, in the present invention, it is generally more desirable to design the expression of thioredoxin and / or thioredoxin reductase in seeds for accumulation and inactivity during grain development. Several strategies are employed to create seeds that express thioredoxin and / or transgenic thioredoxin reductase without having a significant impact on the normal development of the seeds, for example: (i) To share the thioredoxin or thioredoxin reductase active, in such a way which does not interact in a significant way with the target proteins, for example, by the direction to, or the expression in, amyloplasts. Plastid targeting sequences are used to direct accumulation in the amyloplast. In an alternative way, thioredoxin and / or thioredoxin reductase is directed towards an extracellular location in the cell walls, using secretion signals. Finally, in the case of monocotyledons, the expression is used in cell types such as aleurone during the development of the seeds, to keep thioredoxin and / or thioredoxin reductase away from the storage components of the rest of the endosperm. (ii) To design the expression of thioredoxin and / or thioredoxin reductase from thermophilic organisms. Enzymes that have little or no activity at ambient temperatures (as high as 38 ° C to 39 ° C in the field) are less likely to cause problems during development. Preferably, therefore, the enzymes are active primarily at high temperatures, for example, temperatures greater than 40 ° C, more preferably 45 ° C to 60 ° C for wet milling, or still higher, for example 45 ° C. C at 95 ° C. (iii) To place thioredoxin and / or thioredoxin reductase under the control of an inducible promoter, for example, a chemically inducible promoter, a wound-inducible promoter, or a transactivator-regulated promoter that is activated after pollination by a plant that expresses the transactivator. (iv) To use thioredoxin that has specific requirements for a particular thioredoxin reductase, in such a way that the thioredoxin or thioredoxin reductase activity is adequately regulated by means of the availability of thioredoxin reductase or appropriate thioredoxin, respectively. For example, thioredoxin and thioredoxin reductase are expressed in different plants, such that the active combination is only available in the seeds after pollination by the plant expressing the complementary enzyme. Alternatively, thioredoxin or thioredoxin reductase is sequestered in the cell, for example, in a plastid, vacuole, or apoplast, as described above, so that it does not become available until the grain is processed . Grain Processing Methods Accordingly, the invention provides a novel method for improving the separation of starch from the protein matrix, using thioredoxin and / or thioredoxin reductase. In a first embodiment, thioredoxin activity is found to be useful in a variety of seed processing applications, including wet milling, dry milling, oil seed processing, soy bean processing, wheat processing, and wheat quality. flour / dough, more especially the wet milling of grains, in particular maize. According to the foregoing, the invention provides a method for • improving grinding efficiency or increasing grinding performance, • for increasing the separation efficiency of starch and protein, • for improving the yields of soluble proteins at from the grain, or • to increase the solubility of the protein in water or other solvents which comprises impregnating the grain in the presence of complementary thioredoxin and / or thioredoxin, and separating the starch and protein components from the grain. Normally, the impregnation occurs before grinding, but may occur afterwards, and there may be more than one step of grinding or impregnation in the extraction of the process method, and a higher yield of protein from the seeds during the impregnation or after impregnation. Preferably, thioredoxin and / or complementary thioredoxin reductase is provided by the expression of a transgene in the plant from which the grain is harvested. The invention further provides: the use of thioredoxin or thioredoxin reductase in a method for improving milling efficiency, or for increasing the milling performance of starches or proteins, for example, in any of the methods described above, impregnation water comprising an amount of thioredoxin and / or thioredoxin reductase effective to facilitate the separation of the starch from the protein in the grain, • grain that has been exposed to thioredoxin in an amount effective to facilitate separation of the starch from the protein; and • starch or protein that has been produced by the method described above. The thioredoxin activity in the above method can be improved by supplementing the impregnation water with thioredoxin reductase and / or NADPH. Other components normally present in the impregnation water for wet milling may also be present, such as bacteria that produce lactic acid. Preferably, the impregnation is carried out at a temperature of about 52 ° C for a period of 22 to 50 hours, so that it is desirable that the thioredoxin be stable under these conditions. The grain may be a dicotyledonous seed, for example an oil seed, for example, soybeans, sunflower or sugarcane, preferably soybeans; or it can be a monocot seed, for example a cereal seed, for example corn, wheat, oats, barley, rye, or rice, more preferably maize. Thioredoxin can be any protein carrying thiol groups, which can be reversibly oxidized to form disulfide bonds, and can be reduced by NADPH, in the presence of a thioredoxin reductase. Preferably, thioredoxin is derived from a thermophilic organism, as described above. Thioredoxin and / or thioredoxin reductase for use in the present invention is suitably produced in a designed microbe, for example a yeast or Aspergi llus, or in a designed plant capable of very high expression, for example in barley, for example under the control of an active promoter during malt formation, such as a p-alpha-amylase promoter or other gibberellin-dependent promoters. The thioredoxin is then added (in an excreted or extracted form, or in combination with a producer organism or parts thereof) to the impregnation water. As an alternative or complement to the addition of thioredoxin to the impregnation water, the enzyme can be expressed directly in the seed to be ground. Preferably, the enzyme is expressed during the maturation of the grain, or during a conditioning process. According to the foregoing, in a further embodiment, the invention provides: • a method for making thioredoxin on an industrial scale in a transgenic organism, for example a plant, for example a cereal, such as barley or corn, or a microorganism, for example a yeast or Aspergillus, for example a method comprising the steps of culturing a transgenic organism having a chimeric gene expressing thioredoxin, and optionally isolating or extracting thioredoxin, • a method for using transgenic plants that produce high amounts of thioredoxin during maturation or germination of the seed, in such a manner that the quality of the proteins in that seed be affected by thioredoxin endogenously synthesized during seed development, or during the impregnation process, eliminating or reducing in this way the need to condition with exogenous chemicals or enzymes before grinding , • a method to make transgenic plants that produce high amounts of t iorredoxin during the maturation or germination of the seed, in such a way that the quality of the proteins in that seed is affected by the thioredoxin during the development of the seed, or during the impregnation process, eliminating or reducing in this way the need for conditioning with exogenous chemicals or enzymes before grinding, • a method for grinding grain using transgenic seeds containing thioredoxin, which results in higher yields of starch and soluble protein Expression of thioredoxin and thioredoxin reductase in transgenic organisms The invention further comprises a transgenic organism having in its genome a chimeric expression cassette comprising a coding region encoding a thioredoxin or thioredoxin reductase thermostable under the operative control of a promoter. . Preferably, the transgenic organism is a plant that expresses thioredoxin and / or thioredoxin reductase in a form that does not occur naturally in the plants of that species or that expresses thioredoxin at levels higher than what occurs naturally in a plant of that species. Preferably, the thioredoxin is expressed in the seeds during the development of the seeds, and consequently, it is preferably under the control of a seed-specific promoter. Optionally, the expression of thioredoxin is placed under the control of an inducible promoter or regulated by trans activator, so that expression is activated by chemical induction or hybridization with a transactivator when desired. Thioredoxin is suitably directed to the vacuoles of the plant by fusion with a targeting sequence to the vacuole. In the present invention, the thioredoxin coding sequences are fused with the active promoters in the plants, and are transformed into the nuclear genome or the plastid genome. The promoter of preference is a seed-specific promoter, such as the gamazein promoter. The promoter may alternatively be a chemically inducible promoter, such as the tobacco PR-la promoter; or it can be a promoter regulated by chemically inducible transactivator, wherein the transactivator is under the control of a chemically induced promoter; however, in certain situations, constitutive promoters can be used, such as the 35S promoter of CaMV or the Gelvin promoter. With a chemically inducible promoter, the expression of the thioredoxin genes transformed in plants can be activated at an appropriate time by foliar application of a chemical inducer. In an alternative manner, the thioredoxin coding sequence is under the control of a promoter regulated by the transactivator, and expression is achieved by crossing the transformed plant with this sequence, with a second plant expressing the transactivator. In a preferred form of this method, the first plant containing the thioredoxin coding sequence, is the seed progenitor and has male sterility, while the second plant that expresses the transactivator, is the pollinator. The expression of thioredoxin in the seeds is achieved by interplanting the first and second plants, for example, in such a way that the first plant is pollinated by the second, and the thioredoxin is expressed in the seeds of the first plant by the activation of the regulated promoter. by transactivator, with the transactivator expressed by the transactivator gene of the second progenitor. Accordingly, the invention provides a plant that expresses a thioredoxin and / or reductase of thioredoxin, for example, a thioredoxin and / or thioredoxin reductase that is 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 the control of an inducible promoter, for example a chemically inducible promoter, for example operably linked to the inducible promoter or under the control of the promoter regulated by transactivator, wherein the corresponding transactivator is under the 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; also including this plant seeds for the same, whose seeds optionally are treated (for example, they are primed or coated) and / or packed, for example they are placed in a bag with instructions for their use, and the seeds are harvested from the same, for example, to be used in a grinding process as described above. The transgenic plant of the invention optionally may further comprise genes for a better production of thioredoxin reductase and / or NADPH.

The invention further provides: • a method for producing thioredoxin, which comprises culturing a plant that expresses thioredoxin as described above, • a method for producing starch and / or protein, which comprises extracting starch or protein from seeds harvested from a plant as described above; and • a method for wet milling, which comprises impregnating the seeds of a plant expressing thioredoxin as described above, and extracting starch and / or protein therefrom.

The invention further provides: an expression cassette expressible in plants, which comprises a coding region for a thioredoxin or thioredoxin reductase, preferably a thioredoxin derived from a thermophilic organism, for example an archaea, for example from M. ja nna s ch ii or A. ful gi dus, for example as described above, wherein the coding region is preferably optimized to contain codons preferred by the plant, this coding region being operably linked with promoter and terminator sequences that function in a plant, wherein the preferably promoter is a seed-specific promoter or an inducible promoter, for example a promoter chemically inducible or regulated by transactivator; for example an expression cassette expressible in the plastid or in the nucleus comprising a promoter, for example, a regulated promoter or transactivator, regulated by a nuclear transactivator (for example, the T7 promoter when the transactivator is T7 RNA polymerase, whose expression is optionally under the control of an inducible promoter), a vector comprising this expression cassette expressible in plants, a plant transformed with this vector; or • a transgenic plant comprising in its genome, for example, its nuclear or plastid genome, this expression cassette expressible in plants. The invention also comprises a method for producing grain comprising hlevels of thioredoxin or thioredoxin reductase, which comprises pollinating a first plant comprising a cassette of heterologous expression comprising a romoter regulated by transactivator, regulated and operatively linked to a sequence of DNA that codes for a thioredoxin or thioredoxin reductase, being the first plant preferably emasculated or of male sterility; with pollen from a second plant comprising a cassette of heterologous expression comprising a promoter operably linked to a DNA sequence encoding a transactivator capable of regulating the promoter regulated by transactivator; recover the grain of the plant so pollinated.

DEFINITIONS In order to ensure a clear and consistent understanding of the specification and the claims, the following definitions are provided: "expression cassette", as used herein, means a DNA sequence that can direct the expression of a gene in plant cells, which comprises a promoter operably linked to a coding region of interest, which is operatively linked to a termination region. The coding region normally encodes a protein of interest, but it can also code for a functional RNA of interest, for example antisense RNA, or an untranslated RNA which, in the sense or anti-sense direction, inhibits the expression of a particular gene, for example anti-sense RNA. The gene can be chimeric, meaning that at least one omponent of the gene is heterologous with respect to at least one other component of the gene. The gene may also be one that occurs naturally, but has been obtained in a recombinant form useful for the genetic transformation of a plant. However, normally the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell, and must have been introduced into the host cell or an ancestor of the host cell through a transformation event. A "cassette of nuclear expression" is an expression cassette that is integrated into the host's nuclear DNA. A "plastic expression cassette" is an expression cassette that is integrated into the host plastid DNA. A plastid expression cassette as described herein, may optionally comprise a polycistronic operon containing two or more cistronic coding sequences of interest under the control of a single promoter, for example a promoter regulated by transactivator, for example, in wherein one of the coding sequences of interest codes for an anti-sense mRNA, which inhibits the expression of cIpP or other plastid protease, thereby improving the accumulation of protein expressed in the other coding sequence or sequences of interest. * Heterologist ", as used herein, means" of different natural origin. "For example, if a plant is transformed with a gene derived from another organism, particularly from another species, that gene is heterologous with respect to that plant, and also with respect to the descendants of the plant carrying that gene. "Homoplast idico" refers to a plant, plant tissue, or plant cell, where all the plastids are genetically identical, this is the normal state in a When plastids have not been transformed, mutated, or otherwise genetically altered plastids In different tissues or stages of development, plastids can take different forms, for example chloroplasts, own tissues, etioplastos, amiloplasts, cromoplas coughs, and so on. An "inducible promoter" is a promoter that initiates transcription only when the plant is exposed to some particular external stimulus, as distinguished from onstitutiv promoters. or the specific promoters for a specific tissue or organ or stage of development. Particularly preferred inducible promoters for the present invention are promoters chemically inducible or regulated by transactivator. Chemically inducible promoters include plant derived promoters, such as promoters of the acquired systemic resistance pathway, for example PR promoters, for example the PR-1, PR-2, PR-3, PR-4, and PR promoters. -5, especially the PR-la promoter of tobacco and PR-1 promoter of Arabi dopsi s, which initiate transcription when exposed to BTH plant and related chemical products. See U.S. Patent No. 5,614,395, incorporated herein by reference, and International Publication Number WO 98/03536, incorporated herein by reference. Chemically inducible promoters also include receptor-mediated systems, for example those derived from other organisms, such as steroid-dependent aethetic expression, copper-dependent gene expression, tetracycline-dependent gene expression, and particularly the expression system used the USP receptor of Dros oph ila mediated by juvenile growth hormone and its agonists, described in PCT Publication Number 96/04224, incorporated herein by reference, as well as systems using combinations of receptors, for example as described in Publication Number PCT 96/00686, incorporated herein by reference. Chemically inducible promoters can be directly linked to the thioredoxin gene, or the thioredoxin gene can be under the control of a promoter regulated by transactivator, while the gene for the transactivator is under the control of a chemically inducible promoter. See in general C. Gatz, "Chemical Control of Gene Expression", Annu. Rev. Plant Physiol. Plant Mol. Biol. (1997), 48: 89-108, the content of which is incorporated herein by reference. Promoters regulated by transactivator are described more fully later, and can also be induced by the hybridization of a plant comprising the thioredoxin gene under the control of a promoter regulated by transactivator, with a second plant expressing the transactivator. An "isolated DNA molecule" is a nucleotide sequence that, by the hand of man, exists apart from its native environment, and therefore, is not a product of nature. An isolated nucleotide sequence can exist in a purified form, or it can exist in a non-native environment, such as, for example, a cell. transgenic host. A "protein", as defined herein, is the entire protein encoded by the corresponding nucleotide sequence, or is a portion of the protein encoded by the corresponding portion of the nucleotide sequence. An "isolated protein" is a protein that is encoded by an isolated nucleotide sequence, and therefore, is not a product of nature. An isolated protein can exist in a purified form, or it can exist in a non-native environment, such as a transgenic host cell, where the protein is normally not expressed, expressed in a different form or in a different amount in a cell non-isogenic non-transgenic host. A "plant" refers to any plant or part of a plant at any stage of development, and is intended to specifically cover plant and plant material that has been damaged, crushed, or destroyed, as well as viable plants, cuttings, crops of cells or tissues, and seeds. "DNA mixture" is a method for introducing mutations or reconfigurations, preferentially in a random manner, into a DNA molecule, or for generating exchanges of DNA sequences between two or more DNA molecules, preferably in a random manner. The DNA molecule resulting from the mixture of DNA which is a mixed DNA molecule which is a DNA molecule that does not occur naturally, derived, from at least one template DNA molecule. The mixed DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has a biological activity altered with respect to the enzyme encoded by the template DNA. In its broadest sense, the term "substantially similar", when used herein with respect to a nucleolide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide which has substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, for example when only changes occur in amino acids that do not affect the function of the polypeptide. Desirably, the substantially similar nucleotide sequence encodes the polypeptide encoding the reference nucleotide sequence. The percent identity between the substantially similar nucleotide sequence and the reference nucleotide sequence is desirably at least 80 percent, more desirably at least 85 percent, preferably at least 90 percent, more preferably at least 95 percent, still more preferably at least 99 percent. Sequence comparisons are made using a Smith-Waterman sequence alignment algorithm (see, for example, ..ateririan, M.S. Introduction to Computational Biology: Maps, Sequences and Genomes.; Hall. London: 1995, ISBN 0- 12-99391-0, or at http: // wwh ^ hto .usc.edu / software / sepaln / index.html). The locáis program, version 1.16, is used with the following parameters: coupling: 1, fine for poor coupling: 0.33, fine for open hole: 2, fine for extended gap: 2. A nucleotide sequence "substantially similar" to the sequence of reference nucleotides, hybridizes to the reference nucleotide sequence in 7 percent sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA, at 50 ° C, washed in 2X SSC, 0.1 SDS one hundred to 50 ° C, more desirably in 7 percent sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50 ° C, with washing in IX SSC, 0.1 percent SDS at 50 ° C, more Desirably still in 7 percent sodium dodecyl sulphate (SDS), 0.5 M NaP04, 1 mM EDTA at 50 ° C, with 0.5X SSC wash, 0.1 to 50 ° C SDS, preferably in sodium dodecyl sulfate 7 percent (SDS), 0.5 M NaP04, 1 mM EDTA at 50 ° C, with washing at 0. IX SSC, 0.1 percent SDS at 50 ° C, more preferably in dodecyl sulfate co-sodium 7 percent (SDS), NaP04 0.5 M, EDTA 1 mM at 50 ° C, washed in 0.1X SSC, 0.1 percent SDS at 65 ° C. The term "substantially similar", when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. when only changes occur in the amino acids that do not affect the function of the polypeptide. When used for a protein or amino acid sequence, the percent identity between the protein or amino acid sequence substantially similar to that of reference, desirably is at least 80 percent, more desirably 85 percent, preferably at least 90 percent, more preferably at least 95 percent, and still more preferably at least 99 percent. A "transactivator" is a protein that, by itself or in combination with one or more additional proteins, is capable of causing the transcription of a coding region under the control of a promoter regulated by corresponding transactivator. Examples of transactivator systems include the T7 phage promoter of gene 10, whose transcription activation depends on a specific RNA polymerase, such as the RNA polymerase of phage T7. The transactivator is usually an RNA polymerase or a DNA binding protein capable of interacting with a particular promoter to initiate transcription, either by activating the promoter directly, or by inactivating a repressor gene, for example by suppression of the expression or accumulation of a repressor protein. The DNA binding protein can be a chimeric protein comprising a binding region (e.g., the GAL4 binding region) linked to an appropriate transcription activator domain. Some transactivator systems may have multiple transactivators, for example promoters that require not only a polymerase, but also a specific subunit (sigma factor) for promoter recognition, DNA binding, or transcription activation. The transactivator is preferably heterothous with respect to the plant.

Modification of Microbial Genes to Optimize Nuclear Expression in Plants If desired, the cloned thioredoxin genes described in this application can be modified for expression in transgenic host plants. For example, transgenic expression in gene plants derived from microbial sources may require the modification of these genes to achieve and optimize their expression in plants. In particular, bacterial open reading frames that encode separate enzymes, but which are encoded by the same transcript in the native microbe, are best expressed in plants on separate transcripts. To accomplish this, each microbial open reading frame is individually isolated, and cloned into a cassette that provides a plant promoter sequence at the 5 'end of the open reading frame, and a plant transcription terminator at the 3 end. 'of the open reading frame. The sequence of the open reading frame isolated preferably includes the start codon ATG and the stop codon STOP, but may include an additional sequence beyond the start codon ATG and the codon STOP. In addition, the open reading frame may be truncated, but still retain the required activity; for particularly long open reading frames, truncated versions that retain activity for expression in transgenic organisms may be preferable. "Plant promoter" and "plant transcription terminator" mean transcription promoters and terminators that operate inside plant cells. This includes transcription promoters and terminators that can be derived from sources other than plants, such as viruses (examples include promoters and terminators derived from the Cauliflower Mosaic Virus, or from the opine synthase genes in the Ti or Ri plasmids of Agroba ct eri um). In some cases, modification to the coding sequences of the open reading frame and the adjacent sequence will not be required, in which case, it is sufficient to isolate a fragment containing the open reading frame of interest, and insert it downstream of a promoter. of plant. However, preferably, the adjacent microbial sequences that are attached upstream of the ATG, and downstream of the STOP codon, should be minimized or eliminated. In practice, this construction may depend on the availability of the restriction sites.

In other cases, the expression of genes derived from microbial sources can provide problems in expression. These problems have been well characterized in the art, and are particularly common with genes derived from certain sources, such as Ba ci l l us. Modification of these genes can be undertaken using techniques well known in the art. The following problems are typical of those that can be found: 1. Use of Codons The use of preferred codons in the plant differs from the use of preferred codons in certain microorganisms. The comparison of the use of codons within a cloned microbial open reading frame, with the use in genes of plants (and in particular genes of the target plant), will make possible an identification of the codons within the frame, of open reading that should change preference Normally, plant evolution has tended toward a strong preference for nucleotides C and G at the third base position of monocots, while dicotyledons often use nucleotides A or T in this position. By wording a gene to incorporate the use of preferred codons for a particular target transgenic species, many of the problems described below will be overcome for GC / AT content and illegitimate splicing. 2. GC / AT content Plant genes normally have a GC content of more than 35 percent. The sequences of open reading frames that are rich in nucleotides A and T can cause several problems in these plants. First, it is believed that ATTTA motives cause destabilization of messages, and are found at the 3 'end of many short-lived mRNAs. Second, the presentation of polyadenylation signals, such as AATAAA in inappropriate positions within the message, is believed to cause premature truncation of transcription. In addition, monocotyledons can recognize AT-rich sequences as splice sites (see below). 3. Sequences Adjacent to the Start Methionine Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Rather, it is believed that ribosomes bind to the 5 'end of the message, and look for the first available ATG to start the translation. However, it is believed that there is a preference for certain nucleotides adjacent to the ATG, and that the expression of the microbial genes can be improved by the inclusion of a eukaryotic consensus translation initiator in the ATG. Clontech (1993/1994 catalog, page 210) has suggested the sequence GTCGACCAATGGTC (SEQ ID NO: 8) as a consensus translation primer for the expression of the ui dA gene of E. Col i in plants. In addition, Joshi (NAR _15_: 6643-6653 (1987)) has compared many sequences of plants adjacent to the ATG, and suggests the consensus i TAAACAATGGCT (SEQ ID NO: 9). In situations where difficulties are encountered in the expression of open microbial reading frames in plants, the inclusion of one of these Sequences in the initial ATG can improve translation. In such cases, the last three nucleotides of the consensus may not be appropriate to be included in the modified sequence, due to their modification of the second AA residue. Preferred sequences adjacent to the starting methionine may differ between different plant species. A study of 14 maize genes located in the GenBank database provided the following results: Position before start ATG in 14 Maize Genes - 1 0 - 9 - 8 - 7 -6 -4 -3 -2 C 3 8 4 6 0 10 7 T 3 or 3 4 O A 2 3 1 4 3 This analysis can be done for the desired plant species in which thioredoxin or thioredoxine eductase genes are being incorporated, and the sequence adjacent to the ATG can be modified to incorporate the preferred nucleotides. . Removal of Illegitimate Splicing Sites Genes cloned from sources other than plants, and not optimized for expression in plants, may also contain motifs that can be recognized in plants as the 5 'or 3' splice sites, and can be dissociated, thus generating truncated or deleted messages. These sites can be removed using the techniques described in Patent Application Number WO 97/02352, incorporated herein by reference. Techniques for modifying coding sequences and adjacent sequences are well known in the art. In cases where the initial expression of a microbial open reading frame is low, and it is considered appropriate to make alterations to the sequence as described above, then the construction of synthetic genes can be performed according to the well-known methods in this countryside. These, for example, are disclosed in published patent disclosures EP 0,385,962, EP 0,359,472, and WO 93/07278. In most cases, it is preferable to assay the expression of the genetic constructs using transient assay protocols (which are well known in the art) before they are transferred to the transgenic plants. A major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification. Codon adaptation, et cetera, as described above is not required, and plastids are capable of expressing multiple open reading frames under the control of a single promoter. Construction of Transformational Vectors of Selectable Plants and Markers Numerous transformation vectors are available for the transformation of plants, and the genes of this invention can be used in conjunction with any of these vectors. The selection of the vector to be used will depend on the preferred transformation technique and on the tt species for the transformation. For certain tt species, different antibiotic or herbicide selection markers may be preferred. The selection markers routinely used in the transformation include the np tll gene that confers resistance to kanamycin and related antibiotics (Vieira and Messing, Gene 1 9_: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983)), the ba r gene that confers resistance to the herbicide foSph lno t ri ci na (White et al., Nucí Acids Res _18_: 1062 (1990), Spencer et al., Theor Genet 7_9: 625-631 (1990)) , the hp t gene, which confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol CelL Biol 4_2929-2931), the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J.2 (7): 1099- 1104 (1983)); and the mannosa-6-phosphate isomerase gene, which confers the ability to metabolize the mannose, as described in U.S. Patent No. US 5767378. Requirements for the Construction of Expression Cassettes in Plants The genetic sequences intended for expression in transgenic plants, they are first assembled into expression cassettes behind a suitable promoter and upstream of a suitable transcription terminator. These expression cassettes can then be easily transferred to the plant transformation vectors described above. 1. Selection of the Promoter The selection of the promoter used in the expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. The selected promoters will express the transgenes in specific cell types (such as leaf epidermal cells)., mesophilic cells, cells of the cortex of the root), or in specific tissues and organs (roots, seeds, leaves, or flowers, for example), and this selection will reflect the desired location of thioredoxin biosynthesis. In the present invention, seed-specific promoters are preferred. In an alternative way, the selected promoter can drive the expression of the gene under a light-induced promoter or other temporarily regulated promoter. A further alternative is that the selected promoter be inducible by an external stimulus, for example the application of a specific chemical inducer, or by hybridization with a second plant line, which provides the possibility of inducing the transcription of thioredoxin only when desired. 2. Transcription Terminators A variety of transcription terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Suitable transcription terminators and those known to work in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the rbcs E9 pea terminator. These can be used in both monocots and dicots. 3. Sequences for Melting or Regulating Expression Numerous sequences have been found to improve gene expression from within the transcription unit, and these sequences can be used in conjunction with the genes of the invention to increase their expression in transgenic plants. It has been shown that different introns sequences improve expression, particularly in monocotyledonous cells. For example, it has been found that the introns of the Adh l gene of maize significantly improve the expression of a wild-type gene under its known promoter when introduced into maize cells. Intron 1 was found to be particularly effective and improved expression in fusion constructs with the acet-il-trans-transferase gene of chloramphenicol (Callis et al., Genes Develop, 1183-1200 (1987)). In the same experimental system, the intron of the bronz gene of corn had a similar effect to improve expression. Beha have routinely incorporated introns sequences into plant transformation vectors, usually within the non-translated leader. It is also known that a number of untranslated leader sequences derived from viruses improve expression, and these are particularly effective in dicotyledonous cells. Specifically, it has been shown that the leading sequences of Tobacco Mosaic Virus (TMV, the "O-sequence"), Corn Chlorotic Speck Virus (MCMV), and Alfalfa Mosaic Virus (AMV), are effective in improving expression (for example Gallie et al., Nucí Acids Res. ^ 5: 8693-8711 (1987); Skuzeski et al., Plant Molec. Biol. 15_: 65-79 (1990)). 4. Direction of the Genetic Product to Inside of the Cell It is known that there are different mechanisms to direct the genetic products in plants, and the sequences that control the functioning of these mechanisms have been characterized in some detail. The amino-terminal sequences are responsible for the direction to the endoplasmic reticulum, the apoplastoi and the extracellular secretion from the aleurone cells (Koehler and Ho, Plant Cell 2 2: 769-783 (1990)). Additionally, the amino-terminal sequences, in conjunction with the carboxy-terminal sequences, are responsible for the vacuolar direction of the gene products (Shinshi et al., Plant Molec, Biol. LM: 357-368 (1990)). By fusing the appropriate targeting sequences described above to the transgenic sequences of interest, it is possible to direct the transgene product to any organelle or cell compartment. The selected signal sequence must include the known dissociation site, and the constructed fusion must take into account any amino acids after the dissociation site, which are required for dissociation. In some cases, this requirement can be met by the addition of a small number of amino acids between the dissociation site and the ATG of the transgene, or alternatively the replacement of some amino acids within the sequence of the transgene.

The above-described mechanisms for cell targeting can be used not only in conjunction with their known promoters, but also in conjunction with heterologous promoters, to effect a specific cell targeting goal under the transcription regulation of a promoter having a standard of expression different from that of the promoter from which the directional signal is derived. Examples of Expression Cassette Construction The present invention encompasses the expression of thioredoxin genes under the regulation of any promoter that can be expressed in plants, regardless of the origin of the promoter. In addition, the invention encompasses the use of any plant-expressible promoter in conjunction with any additional sequences required or selected for the expression of the thioredoxin or thioredoxin reductase gene. These sequences include, but are not restricted to, transcription terminators, foreign sequences to improve expression (such as introns [e.g. Adh 1 intron], viral sequences [e.g., TMV-O], and sequences intended to direct the Genetic product to specific cell organelles and compartments Different chemical regulators may be employed to induce the expression of the thioredoxin or thioredoxin reductase coding sequence in transformed plants according to the present invention. "Chemical regulators" include chemicals that are known to be inducers for the PR-la promoter in plants (described in U.S. Patent No. 5,614,395), or narrow derivatives thereof A preferred group of regulators for the chemically inducible genes of this invention, is based on the structure of benzo-1, 2, 3- thiadiazole (BTH), and includes, but is not limited to, the following types of compounds: benzole acid, 2,3-thiadiazolecarboxylic acid, benzo-1,2,3-thiadiazolethiocarboxylic acid, cyanobenzo-1,2,3-thiadiazole , benzo-1,2,3-thiadiazolecarboxylic acid amide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide, benzo-1,2,3-thiadiazole 7-carboxylic acid, benzo-1,2,3 acid -thiadiazole-7-thiocarboxylic acid, 7-cyano-benzo-l, 2,3-thiadiazole, benzo-1,2,3-thiadiazole-7-carboxylic acid amide, benzo- 1, 2, 3- hydrazide thiadiazole-7-carboxyl ico, benzo-1,2,3-thiadiazolecarboxylate alkyl, wherein the alkyl group contains 1 to 6 carbon atoms, benzoyl, 2,3-thiadiazole-7-carboxylic acid methyl , benzo-1, 2, 3-thiadiazole-7-carboxylic acid propyl normal, benzo-1,2,3-thiadiazole-carboxylic acid benzyl ester, secondary butyl hydrazide of benzo-1,2,3-thiadiazole-7 carboxyl, and its appropriate derivatives. Other chemical inducers may include, for example, benzoic acid, salicylic acid (SA), polyacrylic acid and its substituted derivatives; Suitable substituents include lower alkyl, lower alkoxy, lower thioalkyl, and halogen. Still another group of regulators for the chemically inducible DNA sequences of this invention is based on the carboxylic acid structure of pyridine, such as the isonic acid structure, and preferably the structure of the haloisonicotonic acid. Dichloroisonicic acids and their derivatives are preferred, for example, lower alkyl esters. Suitable regulators of this class of compounds are, for example, 2,6-dichloroisonicotonic acid (INA), and its lower alkyl esters, especially the methyl ester. Constitutive Expression: the Actin Promoter It is known that several isoforms of actin are expressed in most cell types, and consequently, the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the Actl rice gene has been cloned and characterized (McElroy et al., Plant Cell 2: 163-171 (1990)). It was found that a 1.3 kb fragment of the promoter contains all the regulatory elements required for expression in rice protoplasts. In addition, numerous expression vectors based on the Ac tl promoter have been specifically constructed for use in monocots (McElroy et al., Mol.Gennet Genet. 231: 150-160 (1991)). These incorporate the Ac tl -1 intron, the Adhl 5 'flanking sequence, and the Adhl-1 intron (from the maize alcohol dehydrogenase gene), and the sequence from the CaMV 35S promoter. The vectors that showed the highest expression were 35S fusions and the Actl intron, or the flanking sequence 5 'Ac tl and the intron Ac tl. Optimization of the sequences around the starting ATG (or the GUS reporter gene) also improved expression. The expression cassettes of the promoter described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for the expression of the genes of the invention, and are particularly suitable for use in monocotyledonous hosts. . For example, fragments containing the promoter can be removed from the McElroy constructs, and can be used to replace the double 35S promoter in pCGN1761ENX, which is then available for insertion or for specific genetic sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, it has also been found that the Ac t l rice promoter, with its first intron, directs high expression in cultured barley cells (Chibbar et al., Plant Cell Rep. L_2: 506-509 (1993)). Constitutive Expression: the Ubiquitin Promoter Ubiquitin is another genetic product that is known to accumulate in many cell types, and its promoter has been cloned from several species for use in transgenic plants (eg, sunflower-Binet et al. , Plant Science 79: 87-94 (1991), corn - Christensen et al., Plant Molec.

Biol l_2: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic monocotyledonous systems, and its sequence and vectors constructed for the transformation of monocotyledons are disclosed in Patent Publication Number EP 0,342,926. In addition, Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) comprising the maize ubiquitin promoter, and the first intron, and its high activity in cell suspensions of numerous monocotyledons. when it is introduced by means of microprojectile bombardment. The ubiquitin promoter is suitable for the expression of thioredoxin genes in transgenic plants, especially monocotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter, and / or introns sequences.

Root-specific Expression Another desirable expression pattern for the thioredoxin thiorredoxin and reductase of the present invention is root expression, for example, to improve the extraction of starches and sugars from root crops, such as sugar beet and potato. A suitable root promoter is that described by Framond (FEBS 290: 103-106 (1991)), and also in the published patent application EP 0,452,269. This promoter is transferred to a suitable vector, such as pCGN1761ENX for the insertion of a thioredoxin or thioredoxin reductase gene, and the subsequent transfer of the whole promoter-terminator cassette to a transformation vector of interest.

Wound-Induced Promoters Wound-inducible promoters may also be suitable for the expression of thioredoxin genes, which are activated when harvested. Numerous of these promoters have been described (eg, Xu et al., Plant Molec. Biol. 22_: 573-588 (1993), Logemann et al., Plant Cell _1: 151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 2_2: 783-792 (1993), Firek collaborators, Plant Molec. Biol. 2_2: 129-142 (1993), Warner et al., Plant J. 3: 191-201 (1993)), and all are suitable for use with the present invention. Logemann et al. Describe the 5 'upstream sequences of the wunl gene of dicotyledonous potato. Xu et al. Show that a wound-inducible promoter from the dicotyledonous potato (pi n2) is active in the monocotyledonous rice. In addition, Rohrmeier and Lehle describe the cloning of corn Wipl cDNA, which is induced by wound, and which can be used to isolate the known promoter using conventional techniques. In a similar way, Firek et al., And Warner et al., Have described a wound-induced gene from the Aspra gus offi ci nal i monocot monocot, which is expressed at the local sites of injury and invasion of pathogens. Using donation techniques well known in the art, these promoters can be transferred to suitable vectors, can be fused with the thioredoxin genes of this invention, and can be used to express these genes at the wound sites of the plant.

Expression with Address to Plast Chen and Jagendorf (J. Biol. Chem. 268: 2363 2367 (1993)) have described the successful use of a chloroplast transit peptide for the importation of a heterologous transgene. This peptide used is the transit peptide from the rbcS gene of Ni co ti a na pl umba gi ni fol i a (Poulsen et al., Mol Gen. Genet 205: 193-200 (1986)). Using the restriction enzymes Dral and Sph I, or Tsp509I and Sph I, the DNA sequence encoding this transit peptide can be separated from the plasmid prbcS-8B, and can be manipulated for use with any of the constructions described above. . The Dral Sph i fragment extends from -58 in relation to the ATG of rbcS from start to, and including, the first amino acid (also a methionine) of the mature peptide immediately after the import dissociation site, while the Tsp509 I-Sph I fragment extends from -8 relative to the ATG of rbcS from start to end, including, the first amino acid of the mature peptide. Accordingly, these fragments can be appropriately inserted into the polylinker of any selected expression cassette, generating a transcription fusion with the untranslated leader of the chosen promoter (e.g., 35S, PR-la, actin, ubiquitin, etc.), while which makes possible the insertion of a thioredoxin gene or thioredoxin reductase in correct fusion downstream of the transit peptide.

Transformation of Dicotyledonous Transformation techniques for dicotyledons are well known in this field, and include techniques based on Agroba ct eri um, and techniques that do not require Agroba ct eri un. The techniques of Agroba ct eri um involve the recovery of the exogenous genetic material directly by the protoplasts or cells. This can be done by means of PEG-mediated recovery or electroporation, mediated delivery by particle bombardment, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology ^: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case, the transformed cells are regenerated to whole plants using conventional techniques known in the art. The transformation mediated by Agroba ct eri um is a preferred technique for the transformation of dicotyledons, due to its high transformation efficiency, and its wide utility with many different species. The many crop species that can be routinely processed by Agroba ct eri um include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybeans, alfalfa, and poplar, see, for example, Publications Numbers EP 0,317,511 (cotton), EP 0,249,432 (tomato), WO 87/07299 (Bra s if ca), or US 4,795,855 (poplar). Transformation with Agroba ct eri um normally involves the transfer of the binary vector carrying the foreign DNA of interest (eg pCIB200 or pCIB2001), to an appropriate Agroba ct eri um strain, which may depend on the complement of the vi rll genes evaded s by the Agrobacterium host strain, either on a co-resident Ti plasmid, or in a chromosomal manner (for example, strain CIB542 for pCIB20O and pCIB2001 (Uknes et al., Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is made by a triparental coupling procedure using E. coli carrying the recombinant binary vector, an auxiliary E. coli strain carrying a plasmid such as pRK2013, and which can mobilize the recombinant binary vector towards the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen and Willmitzer, Nucí Acids Res. 3 ^: 9877 (1988)). The transformation of the target plant species by recombinant Agrobacterium usually involves the co-cultivation of the Agrobacterium with plant explants, and follows the well-known protocols in this field. The transformed tissue is regenerated on a selectable medium carrying an antibiotic or herbicide resistance marker present between the T-DNA boundaries of the binary plasmid. Dicotyledonous transformation can also be done using biolistics. The particularly preferred method for the transformation of soybeans is described in US Pat. No. 5024944.

Transformation of Monocotyledons The transformation of most monocotyledonous species has also become a routine. Preferred techniques include direct gene transfer to the protoplasts using PEG or electroporation techniques, and bombardment of particles to the callus tissue. Transformations - can be undertaken with a single DNA species, or with multiple DNA species (ie, co-trans formation), and both techniques are suitable for use with this invention. The co-trans formation can have the advantage of avoiding the complex construction of the vector, and of generating transgenic plants with unlinked sites for the gene of interest and the selectable marker, making possible the removal of the selectable marker in subsequent generations., if this is considered desirable. However, a disadvantage of the use of co-transformation is the frequency less than 100 percent with which the separated DNA species are integrated into the genome (Schocher et al., Biotechnology 4_: 1093-1096 1986)). Patent Applications Numbers EP 0,292,435, EP 0,392,225 and WO 93/07278 disclose techniques for the preparation of callus and protoplasts from an elite endometrial line of maize, the transformation of protoplasts using PEG or electroporation, and the regeneration of plants of maize from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for the transformation of a corn line derived from A188 using particle bombardment. . In addition, International Patent Application WO 93/07278, and Koziel et al. (Biotechnology 1 ^: 194-200 (1993)) describe techniques for the transformation of elite inbred maize lines by particle bombardment. This technique uses immature maize embryos 1.5 to 2.5 millimeters long, cut from a corn cob 14 to 15 days after pollination, and a Biolistics PDS-100OO device for bombardment. Corn can also be transformed by Agroba c t eri um, for example using the methods described in Ishida et al., 1996; High efficieney transformation of maize (.Zea mayz L.) mediated by Agroba ct eri um t umefa ci ens, Nature Biotechnology 14, 745-750. Rice transformation can also be undertaken by direct gene transfer techniques, using protoplasts or particle bombardment. Transformation mediated by the protoplast has been described for the Japanese types and the Indi ca types (Zhang et al., Plant Cell Rep 7: 379-384 (1988); Shimamoto et al., Nature 338: 274-277 (1989); Datta; and collaborators, Biotechnology 8: 736-740 (1990)). Both types can also be routinely transformed using particle bombardment (Christou et al., Biotechnology 9 ^: 957-962 (1991)). Patent Application Number EP 0,332,581 describes techniques for the generation, transformation, and regeneration of Pooideae protoplasts. These techniques allow the transformation of Da c tyl i s and wheat. In addition, wheat transformation was described by Vasil et al. (Biotechnology 10: 667-674 (1992)), using particle bombardment to the long-term regenerable callus cells type C, and also by Vasil et al. (Biotechnology l. : 1553-1558 (1993)) Weeks et al. (Plant Physio., 102: 1077-1084 (1993)), using bombardment of immature embryo particles and callus derived from immature embryos. However, a preferred technique for wheat transformation involves the transformation of wheat by bombardment of immature embryo particles, and includes either a high pass in sucrose or high pass in maltose, prior to delivery of the gene. Prior to bombardment, any number of embryos (0.75 to 1 millimeter in length) are coated on an MS medium with 3 percent sucrose (Murashige and Skoog, Physiology Planetarium 15: 473-497 (1962)), and 3 ml 1 We equate / li of 2, -D for the induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, the embryos are removed from the induction medium, and placed on the osmotic (i.e., induction medium with added sucrose or maltose at the desired concentration, usually 15 percent). The embryos are allowed to plasmolize for 2 to 3 hours, and then they are bombarded. It is typical 20 embryos per target plate, although it is not critical. A plasmid carrying the appropriate gene (such as pCIB3064 or pSG35) is precipitated onto gold particles in a micron size using conventional methods. Each embryo plate is fired with the Dupont Biolistics® helium device using a burst pressure of approximately 70 kg / cm2, using a standard 80 mesh. After the bombardment, the embryos are placed back in the dark to recover for approximately 24 hours (still on the osmotic). After 24 hours, the embryos are removed from the osmotic, and placed back on the induction medium, where they remain for approximately 1 month before regeneration. Approximately 1 month later, the embryonic explants with embryogenic callus in development are transferred to the regeneration medium (MS + 1 milligram / liter of NAA, 5 milligrams / liter of GA), which also contains the appropriate selection agent (10 milligrams / liter of Basta in the case of pCIB3064, and 2 milligrams / liter of methotrexate in the case of pS0G35). After about 1 month, the developed shoots are transferred to larger sterile containers known as "GA7s", which contain MS at a half concentration, 2 percent sucrose, and the same concentration of selection agent. Patent Application Number WO 94/13822 describes methods for the transformation of wheat, and is incorporated herein by reference.

Plastid Transformation Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513; 5,545,817, and 5,545,818, all of which are expressly incorporated herein by reference in their entirety; in the PCT Applications Nos. WO 95/16783 and WO 98/11235, which are hereby incorporated by reference in their entirety; and in McBride et al. (1994) Proc. Nati Acad. Sci. USA 91, 7301-7305, which is also incorporated herein by reference in its entirety; The basic technique for chloroplast transformation involves introducing regions of the cloned plastid DNA flanking a selectable marker, together with the gene of interest, into a suitable target tissue, for example using biolistics or protoplast transformation (eg, mediated transformation). by calcium chloride or PEG). The flanking regions of 1 to 1.5 kb, called erection, facilitate homologous recombination with the plastid genome, and therefore, allow the replacement or modification of specific regions of the plastome. Initially, point mutations were used in the chloroplast 16S rRNA, and in the rpsl2 genes that confer resistance to spectinomycin and / or streptomycin, as selectable markers for transformation (Svab., Z. Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad Sci. USA 87, 8526-8530, incorporated herein by reference, Staub, JM and Maliga, P. (1992) Plant Cell 4, 39-45, incorporated herein by reference. reference). This resulted in stable homoplasmic transformations at a frequency of about 1 per cent bombardment of target leaves. The presence of cloning sites between these markers allowed the creation of an address vector for the plastid for the introduction of foreign genes (Staub, JM and Maliqa, P. (1993) EMBO J. 12, 601-606, incorporated into the present as reference). Substantial increases in the frequency of transformation were obtained by replacing the rRNA or recessive rRNA-resistant antibiotic resistance genes with a dominant selectable marker, by coding the bacterial gene aa dA to the desominant enzyme of spectinomycin, aminoglycoside- 3 '-adenyltransferase (Svab, Z., and Maliga P. (1993) Proc. Na ti.Ac d.Sci. E UA 90, 913-917, incorporated herein by reference). Previously, this marker had been successfully used for the high-frequency transformation of the green algae plastid genome Chl amydomona s rei nh ardti i (Goldschmidt-Clemont, M. (1991) Nu ci Aci ds Re s.19, 4083 -4089, incorporated herein by reference). Other selectable markers useful for plastid transformation are known in the art and are encompassed within the scope of the invention. Normally, approximately 15 to 20 cycles of cell division are required following the transformation to reach a homoplasty state. The expression of the plastid, where mediane genes are inserted homologous recombination in all the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous number of copies on expression genes that can easily exceed to 10 percent of the total soluble plant protein. However, these high levels of expression may present potential viability problems, especially during the early growth of the plant and its development. Similar problems arise due to the expression of bioactive enzymes or proteins that can be highly detrimental to the survival of the transgenic plants and therefore, if they are expressed in a constitutive manner, they can not be successfully introduced into the plant genome. Therefore, in one aspect, the present invention has coupled the expression in the nuclear genome of an RNA polymerase of the T7 phage targeting the chloroplast under the control of the chemically inducible PR-la promoter (US Pat. No. US Pat. 5,614,395 incorporated as reference) of tobacco, with a chloroplast reporter transgene regulated by the promoter / terminator sequences of T7 of gene 10. For example, when the plastid transformants homoplasmic to the maternally inherited ui dA gene encoding the reporter of β- glucuronidase (GUS), are pollinated by lines that express the T7 polymerase in the nucleus, obtaining the Fl plants that carry both transgenic constructions, but that do not express the GUS protein. The synthesis of large quantities of enzymatically active GUS is triggered in the plastids of these plants, only after foliar application of the PR-la-inducing compound, benzo (1, 2, 3) thiadiazo-1-7- Smethyl ester carbothio (BTH).

BRIEF DESCRIPTION OF THE SEQUENCES IN THE LIST OF SEQUENCES SEQ ID NO: the thioredoxin protein sequence from Methanococcus j annaschii. SEQ ID NO: 2 thioredoxin protein sequence from Archaeoglobus fulgidus (trx-1). SEQ ID NO: 3 thioredoxin protein sequence from Archaeoglobus fulgidus (trx-2). SEQ ID NOM protein sequence of thioredoxin from Archaeoglobus fulgidus (trx-3). SEQ ID NO: 5 thioredoxin protein sequence from Archaeoglobus fulgidus (trx-4). SEQ ID NO: 6 thioredoxin reductase protein sequence from Methanococcus j annaschii (trxB) SEQ ID NO: 7 thioredoxin reductive protein sequence from Archaeoglobus fulgidus (trxB).

SEQ ID NO: 8 Clontech sequence. SEQ ID NO: 9 sequence of Joshi.

EXAMPLES The invention is further described with reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting, unless otherwise specified.

The conventional recombinant DNA and molecular cloning techniques used herein are well known in the art, and are described for example by Sambrook et al. (1989) Molecular Cloning, and by Ausubel et al. (1994) Current Protocols in Molecular Biology Example 1: Transformation of corn with heat-stable thioredoxin A heat-stable thioredoxin expressing gene is prepared from Me thanococc us j anna schi i, having the sequence MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYI VMENPQ KAMEYGIMAVPTIVINGDVEFIGAPTKEALVEAIKKRL (SEQ ID NO: 1) using codons preferred by corn, as described in U.S. Patent No. 5,625,136, under the control of the seed-specific gamma-zein promoter, and the expression cassette is incorporated between the T-DNA boundaries of the pGIOUP plasmid. The T-DNA of this plasmid contains a bar gene expressible in plants driven by the ubiquitin promoter (Christensen et al., Plant Mol.

Biol. 18: 875-689, 1992), to provide resistance to phosphinothricin. It also contains the GUS gene (β-glucuronidase), with an intron at the N-terminal codon of the coding sequence driven by a chimeric promoter (SMAS) derived from the octopine and mannopine synthase genes (a trimer of the current octopine synthase promoter above, which activates the sequence with a domain of the manno synthase gene, Ni et al., Plant J. 7: 661-676, 1995). This GUS-intron gene expresses GUS activity in plant cells, but not in Agroba c t eri um. Alternatively, heat-stable thioredoxin from Me th a noco cc us j anna s chi i is cloned into plasmid pNOV117, which contains a pmi gene expressible in plants driven by the maize ubiquitin promoter for the selection on mañosa (Christensen et al., 1992, Joersbo et al., 1998). The strain LBA4404 of A is used. t umefa ci ens (pAL4404, pSBl) in these experiments. pAL4404 is a disassembled helper plasmid. pSBl is a broad-range host plasmid that contains a region of homology with pGIGUP and pNOV117, and a Kpnl fragment of 15.2 kb from the virulence region of pTiBo542 (Ishida et al., 1996; High efficieney transformation of maize (Zea mays L.) mediated by Agroba ct eri um t umefa ci s, Nature Biotechnology 14, 745-750). The introduction of the plasmid pGIGUP or pNOV117 by electroporation into LBA4404 (pAL4404, pSBl) results in a cointegration of pGIGUP or pNOV117 and pSBl. The T-DNA of pN0V117 contains a mannose-6-phosphate isomerase gene driven by the ubiquitin promoter, to provide the ability to metabolize the maas, as well as the thioredoxin gene described above. Agroba ct erium is grown for 3 days on YP medium (5 grams / liter of yeast extract, 10 grams / liter of peptone, 5 grams / liter of NaCl, 15 grams / liter of agar, pH of 6.8) supplemented with 50 milligrams / liter of spectinomycin, and 10 milligrams / liter of tetracycline. The bacteria are harvested with a cycle, and are suspended in a liquid N6 medium at a density which is from 109 to 5 * 10 cells / milliliter. Agroba ct erium cells can also be harvested from a night culture in a YP medium, and can be resuspended in a liquid N6 medium. For 1 liter of medium, add: 4 grams of N6 powder salts (Sigma, St. Louis, MO), 30 grams of sucrose, 100 milligrams of mycositol, 2 milligrams of glycine, 1 milligram of thiamin, 0.5 milligrams of HCl of pyrodoxin, 0.5 milligrams of nicotinic acid, 2 milligrams of 2,4-D (from a supply solution [1 milligram / milliliter] made by dissolving 2, 4-D in dilute KOH). Adjust to a pH of 6.0 with 1M KOH, add 3 grams of Gelrite, and autoclave. Immature maize embryos are obtained approximately 10 to 14 days after self-pollination. The immature zygotic embryos are divided between different plaques containing medium capable of inducing and supporting the formation of embryogenic callus in approximately 25 immature embryos per plaque. The immature embryos are inoculated either on the plate or in liquid with Agroba ct erium having a Ti plasmid comprising a selectable marker gene. The immature embryos are coated on callus initiation medium containing silver nitrate (10 milligrams / liter) either before or immediately after inoculation with Agrobacterium. Approximately 25 immature embryos are placed on each plate. From 16 to 72 hours after inoculation, the immature embryos are transferred to the callus initiation medium with silver nitrate and cefotaxime. Silver nitrate and cefotaxime. The selection of the transformed cells is carried out as follows: mañosa is used to select the transformed cells i n vi t ro. This selection can be applied as low as 1 gram / liter, 2 to 20 days after inoculation, and can be maintained for a total of 2 to 12 weeks. The embryogenic callus thus obtained is regenerated in the presence or in the absence of mannose, on a standard regeneration medium. All the plants are tested by the chlorophenol red (CR) test to determine the tolerance to the mañosa. This assay uses pH-sensitive indicator dye to show which cells are growing in the presence of mannose. The cells that grow produce a pH change in the medium, and convert the Red Chlorophenol (CR) indicator to yellow from red. Plants that express tolerance to trusses are easily identified in this test. Positive plants by the CR test are tested by reaction in the polymerase chain to determine the presence of the mannose gene. Plants that are positive for the reaction test in the polymerase chain are analyzed by Southern blot. The regenerated plants are tested for the expression of thioredoxin. The plants are of a normal development. The corn kernel from the progeny plants derived from the highest expression event is tested in a small scale wet milling process, and the possibility of extracting the starch is measured, comparing with the corn of the same genotype. without the thioredoxin transgene. The maize expressing the thioredoxin gene exhibits substantially greater availability of starch in the wet milling process than the untransformed isogenic maize.

Example 2: Transformation of maize with thioredoxin or heat-stable thioredoxin reductase Using the procedures described in Example 1, maize is co-trans formed with thioredoxin and thioredoxin reductase genes from M. j anna schi i, described previously. Both genes are under the control of the seed-specific range-zein promoter. The two genes are linked and placed between the right and left boundaries of the pGIGUP or pNOV117 plasmid, to improve the possibility that both genes are incorporated into the plant chromosome as a single insert. The regenerated plants are assayed for the expression of thioredoxin and thioredoxin reductase. The plants are of a normal development.

The maize grain of the progeny plants derived from the highest expression event is tested in a small scale wet milling process, and the possibility of starch extraction is measured, comparing with the corn of the same genotype without the transgenes of thioredoxin / thioredoxin reductase. Corn that expresses the thioredoxin and thioredoxin reductase genes exhibits substantially greater availability of starch in the wet milling process than nontransgenic isogenic corn. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > Novart'is AG < 120 > Thioredoxin and Grain Processing < 130 > S-30758 / A < 140 > 09 / 213,208 < 141 > December 17, 1998 < 160 > 9 < 170 > Patentln Ver. 2.2 < 210 > 1 < 211 > 85 < 212 > PRT < 213 > Methanococcus jannaschii < 400 > 1 Met Ser Lys Val Lys lie Glu Leu Phe Thr Ser Pro Met Cys Pro His 1 5 10 15 Cys Pro Wing Wing Lys Arg Val Val Glu Wing Val Wing Asn Glu Met Pro 20 25 30 Asp Wing Val Glu Val Glu Tyr lie Asn Val Met Glu Asn Pro Gln Lys 35 40 45 Wing Met Glu Tyr Gly lie Met Wing Val Pro Thr lie Val lie Asn Gly 50 55 60 Asp Val Glu Phe lie Gly Ala Pro Thr Lys Glu Ala Leu Val Glu Ala 65 70 75 80 lie Lys Lys Arg Leu 85 < 210 > 2 < 211 > 119 < 212 > PRT < 213 > Archaeoglobus fulgidus < 400 > 2 Met Pro Met Val Arg Lys Ala Ala Phe Tyr Ala lie Ala Val lie Ser 1 5 10 15 Gly Val Leu Ala Ala Val Val Gly Asn Ala Leu Tyr His Asn Phe Asn 20 25 30 Be Asp Leu Gly Ala Gln Ala Lys lie Tyr Phe Phe Tyr Ser Asp Ser 35 40 45 Cys Pro His Cys Arg Glu Val Lys Pro Tyr Val Glu Glu Phe Ala Lys 50 55 60 Thr His Asn Leu Thr Trp Cys Asn Val Wing Glu Met Asp Wing Asn Cys 65 70 75 80 Ser Lys lie Wing Gln Glu Phe Gly lie Lys Tyr Val Pro Thr Leu Val 85 90 95 lie Met Asp Glu Glu Wing His Val Phe Val Gly Ser Asp Glu Val Arg 100 105 110 Thr Ala lie Glu Gly Met Lys 115 < 210 > 3 < 211 > 93 < 212 > PRT < 213 > Archaeoglobus fulgidus < 400 > 3 Met Val Phe Thr Ser Lys Tyr Cys Pro Tyr Cys Arg Ala Phe Glu Lys 1 5 10 15 Val Val Glu Arg Leu Met Gly Glu Leu Asn Gly Thr Val Glu Phe Glu 20 25 30 Val Val Asp Val Asp Glu Lys Arg Glu Leu Wing Glu Lys Tyr Glu Val 35 40 45 Leu Met Leu Pro Thr Leu Val Leu Wing Asp Gly Asp Glu Val Leu Gly 50 55 60 Gly Phe Met Gly Phe Wing Asp Tyr Lys Thr Wing Arg Glu Ala lie Leu 65 70 75 80 Glu Gln lie Be Wing Phe Leu Lys Pro Asp Tyr Lys Asn 85 90 < 210 > 4 < 211 > 134 < 212 > PRT < 213 > Archaeoglobus fulgidus < 400 > 4 Met Asp Glu Leu Glu Leu lie Arg Gln Lys Lys Leu Lys Glu Met Met 1 5 10 15 Gln Lys Met Ser Gly Glu Glu Lys Wing Arg Lys Val Leu Asp Ser Pro 20 25 30 Val Lys Leu Asn Ser Ser Asn Phe Asp Glu Thr Leu Lys Asn Asn Glu 35 40 45 Asn Val Val Asp Phe Trp Wing Glu Trp Cys Met Pro Cys Lys Met 50 55 60 lie Wing Pro Val lie Glu Glu Leu Wing Lys Glu Tyr Wing Gly Lys Val 65 70 75 80 Val Phe Gly Lys Leu Asn Thr Asp Glu Asn Pro Thr lie Wing Wing Arg 85 90 95 Tyr Gly lie Be Ala lie Pro Thr Leu lie Phe Phe Lys Lys Gly Lys 100 105 110 Pro Val Asp Gln Leu Val Gly Wing Met Pro Lys Ser Glu Leu Lys Arg 115 120 125 Trp Val Gln Arg Asn Leu 130 < 210 > 5 < 211 > 105 < 212 > PRT < 213 > Archaeoglobus fulgidus < 400 > 5 Met Glu Arg Leu Asn Ser Glu Arg Phe Arg Glu Val lie Gln Ser Asp 1 5 10 15 Lys Leu Val Val Val Asp Phe Tyr Ala Asp Trp Cys Met Pro Cys Arg 20 25 30 Tyr lie Ser lie lie Leu Glu Lys Leu Ser Lys Glu Tyr Asn Gly Glu 35 40 45 Val Glu Phe Tyr Lys Leu Asn Val Asp Glu Asn Gln Asp Val Ala Phe 50 55 60 Glu Tyr Gly lie Ala Ser lie Pro Thr Val Leu Phe Phe Arg Asn Gly 65 70 75 80 Lys Val Val Gly Gly Phe lie Gly Ala Met Pro Glu Ser Ala Val Arg 85 90 95 Wing Glu lie Glu Lys Wing Leu Gly Wing 100 105 < 210 > 6 < 211 > 301 < 212 > PRT < 213 > Methanococcus jannaschii < 400 > 6 Met lie His Asp Thr lie lie lie Gly Ala Gly Pro Gly Gly Leu Thr 1 5 10 15 Ala Gly lie Tyr Ala Met Arg Gly Lys Leu Asn Ala Leu Cys lie Glu 20 25 30 Lys Glu Asn Wing Gly Gly Arg lie Wing Glu Wing Gly lie Val Glu Asn 35 40 45 Tyr Pro Gly Phe Glu Glu lie Arg Gly Tyr Glu Leu Wing Glu Lys Phe 50 • 55 60 Lys Asn His Wing Glu Lys Phe Lys Leu Pro lie lie Tyr Asp Glu Val 65 70 75 80 lie Lys lie Glu Thr Lys Glu Arg Pro Phe Lys Val lie Thr Lys Asn 85 90 95 Ser Glu Tyr Leu Thr Lys Thr lie Val lie Wing Thr Gly Thr Lys Pro 100 105 110 Lys Lys Leu Gly Leu Asn Glu Asp Lys Phe lie Gly Arg Gly lie Ser 115 120 125 Tyr Cys Thr Met Cys Asp Wing Phe Phe Tyr Leu Asn Lys Glu Val lie 130 135 140 Val lie Gly Arg Asp Thr Pro Ala lie Met Ser Ala lie Asn Leu Lys 145 150 155 160 Asp lie Wing Lys Lys Val lie Val lie Thr Asp Lys Ser Glu Leu Lys 165 170 175 Ala Ala Glu Ser lie Met Leu Asp Lys Leu Lys Glu Ala Asn Asn Val 180 185 190 Glu lie lie Tyr Asn Ala Lys Pro Leu Glu lie Val Gly Glu Glu Arg 195 200 205 Wing Glu Gly Val Lys lie Ser Val Asn Gly Lys Glu Glu lie lie Lys 210 215 220 Wing Asp Gly lie Phe lie Ser Leu Gly His Val Pro Asn Thr Glu Phe 225 230 235 240 Leu Lys Asp Ser Gly lie Glu Leu Asp Lys Lys Gly Phe lie Lys Thr 245 250 255 Asp Glu Asn Cys Arg Thr Asn lie Asp Gly lie Tyr Ala Val Gly Asp 260 265 270 Val Arg Gly Gly Val Met Gln Val Ala Lys Ala Val Gly Asp Gly Cys 275 280 285 Val Ala Met Ala Asn He He Lys Lyr Tyr Leu Gln Lys 290 295 300 < 210 > 7 < 211 > 300 < 212 > PRT < 213 > Archaeoglobus fulgidus < 400 > 7 Met Tyr Asp Val Wing He He Gly Gly Gly Pro Wing Gly Leu Thr Wing 1 5 10 15 Ala Leu Tyr Ser Ala Arg Tyr Gly Leu Lys Thr Val Phe Phe Glu Thr 20 25 30 Val Asp Pro Val Ser Gln Leu Ser Leu Ala Ala Lys He Glu Asn Tyr 35 40 45 Pro Gly Phe Glu Gly Be Gly Met Glu Leu Leu Glu Lys Met Lys Glu 50 55 60 Gln Ala Val Lys Ala Gly Ala Glu Trp Lys Leu Glu Lys Val Glu Arg 65 70 75 80 Val Glu Arg Asn Gly Glu Thr Phe Thr Val He Wing Glu Gly Gly Glu 85 90 95 Tyr Glu Ala Lys Ala He He Val Ala Thr Gly Gly Lys His Lys Glu 100 105 110 Ala Gly He Glu Gly Glu Be Ala Phe He Gly Arg Gly Val Ser Tyr 115 120 125 Cys Ala Thr Cys Asp Gly Asn Phe Phe Arg Gly Lys Lys Val He Val 130 135 140 Tyr Gly Ser Gly Lys Glu Wing He Glu Asp Wing He Tyr Leu His Asp 145 150 155 160 He Gly Cys Glu Val Thr He Val Ser Arg Thr Pro Ser Phe Arg Ala 165 170 175 Glu Lys Ala Leu Val Glu Glu Val Glu Lys Arg Gly He Pro Val His 180 185 190 Tyr Ser Thr Thr He Arg Lys He He Gly Ser Gly Lys Val Glu Lys 195 200 205 Val Val Ala Tyr Asn Arg Glu Lys Lys Glu Glu Phe Glu He Glu Ala 210 215 220 Asp Gly He Phe Val Wing He Gly Met Arg Pro Wing Thr Asp Val Val 225 230 235 240 Wing Glu Leu Gly Val Glu Arg Asp Ser Met Gly Tyr He Lys Val Asp 245 250 255 Lys Glu Gln Arg Thr Asn Val Glu Gly Val Phe Ala Wing Gly Asp Cys 260 265 270 Cys Asp Asn Pro Leu Lys Gln Val Val Thr Ala Cys Gly Asp Gly Ala 275 280 285 Val Ala Ala Tyr Ser Ala Tyr Lys Tyr Leu Thr Ser 290 295 300 < 210 > 8 < 211 > 13 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide < 400 > 8 gtcgaccatg gtc 13 < 210 > 9 < 211 > 12 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide < 400 > 9 taaacaatgg ct 12

Claims (11)

1. CLAIMS The invention having been described as above, the content of the following claims is claimed as property: 1. A method for increasing the efficiency of starch and protein separation in a grain grinding process, characterized in that it comprises impregnating the grain to a elevated temperature in the presence of a thioredoxin and / or complementary thioreoxin reductase and separating the starch and protein components from the grain.
2. 2. A method in accordance with the claim 1, characterized in that the grain includes grain of a transgenic plant, wherein the transgene expresses thioredoxin and / or thioredoxin reductase.
3. 3. A method in accordance with the claim 2, characterized in that the plant is selected from corn. { Zea mays) and soybeans.
4. 4. A plant characterized in that it comprises a heterologous DNA sequence encoding a thioredoxin and / or thioredoxin reductase stably integrated into its nuclear or plastid DNA.
5. 5. The plant according to claim 4, characterized in that thioredoxin and / or reductase of thioredoxin is thermostable.
6. 6. The plant of claim 5, characterized in that thioredoxin and / or reductase of thioredoxin is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NOM, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
7. 7. 7. The plant of any of claims 6, characterized in that the plant is selected from corn and soybeans.
8. 8. An expression cassette expressible in plants, characterized in that it comprises a coding region for a thioredoxin and / or thioredoxin reductase operably linked to promoter and terminator sequences that function in a plant.
9. 9. The expression cassette expressible in plants according to claim 8, characterized in that thioredoxin and / or reductase of thioredoxin is thermostable.
10. 10. The expression cassette expressible in plants of claim 9, characterized in that the thioredoxin and / or reductase of thioredoxin is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NOM, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
11. 11. A method for producing grain, characterized I BEAR OF GRAINS SUMMARY OF THE INVENTION The invention provides novel methods for processing grain, particularly maize and soybeans, using thioredoxin and / or thioredoxin reductase, to improve the possibility of extraction and recovery of starch and protein. The invention further provides novel transgenic plants that express thioredoxin and / or thermostable thioredoxin reductase. fifteen