KR20010108021A - Thioredoxin and grain processing - Google Patents

Abstract

The present invention provides a method for treating cereals, in particular corn and soybeans, using thioredoxin and / or thioredoxin reductase to enhance the extractability and recovery of protein and starch. The present invention also provides novel transgenic plants expressing heat stable thioredoxin and / or thioredoxin reductase.

Description

Thioredoxin and grain processing

The present invention relates, in particular, to novel grain processing methods for enhancing protein and starch recovery in corn wet grinding and soybean processing and to novel transgenic plants useful in such processing.

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 efficiency of grain processing and the quality of products recovered from the grain processing process. The development of effective methods to remove or reduce the degree of binding of protein disulfide bonds in cereals will enhance processing efficiency. In addition, grain behavior in feed is also affected by intermolecular and intramolecular disulfide bonds: lowering the degree of disulfide bonds increases grain digestibility, nutrient bioavailability and neutralization of antitrophic factors such as proteases, amylase inhibitors, etc. You can.

Expression of transformed thioredoxin and / or thioredoxin reductases in corn and soybeans and the use of thioredoxin in grain processing, eg wet grinding, are novel and seed protein during industrial treatment An alternative method for reducing disulfide bonds is provided. Thus, the present invention provides not only grains with modified stock protein quality, but also grains that behave differently in quality from conventional grains during industrial processing or animal digestion (both referred to herein as “treatment”). do. This method of delivering thioredoxin and / or thioredoxin reductase eliminates the need to develop an extraneous source of thioredoxin and / or thioredoxin reductase for addition during processing. A second advantage of supplying thioredoxin and / or thioredoxin reductase through grains is that physical disruption of seed integrity results in contacting the enzyme with the seed storage or matrix protein prior to or as a separate treatment step. It is not necessarily necessary.

Three forms of using thioredoxin in the grain processing process are provided:

(1) expression and action during seed development to modify the composition and quality of harvested grain;

(2) expression during seed development (no activity) to modify the quality of the product during the treatment process; and

(3) Preparation of thioredoxin and / or thioredoxin reductase in grains added during processing to modify the quality of other grain products.

Thus, the present invention provides:

Soaking the grains at elevated temperature in the presence of supplemental thioredoxin and / or thioredoxin reductase, and

A method for enhancing starch and protein separation efficiency in the grain grinding method, including separating starch and protein components of the grain,

The grains mentioned above, wherein the transgene comprises grains from transgenic plants expressing thioredoxin and / or thioredoxin reductase, in particular heat stable thioredoxin and / or thioredoxin reductase. Method and

The method as mentioned above, wherein the plant is selected from dicotyledonous or monocotyledonous plants, in particular cereals, more particularly corn (Zea mays) and soybeans.

The present invention also provides a transgenic plant.

In particular, the present invention provides:

A plant comprising a heterologous DNA sequence encoding thioredoxin and / or thioredoxin reductase, which is stably integrated within the nuclear or chromatin DNA,

-Plants as mentioned above, wherein thioredoxin and / or thioredoxin reductase are thermally stable,

A plant as mentioned above, wherein the thioredoxin and / or thioredoxin reductase is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; and

Plants as mentioned above, selected from corn and soybeans.

In addition, the present invention provides:

An expression cassette expressible in a plant, comprising a coding region for thioredoxin and / or thioredoxin reductase operably linked to a promoter and terminator sequence acting in the plant,

Expression cassettes expressible in plants as mentioned above, in which thioredoxin and / or thioredoxin reductase are heat stable;

-Expressible expression in a plant as mentioned above, wherein the thioredoxin and / or thioredoxin reductase is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 cassette.

In addition, the present invention provides:

A process for the production of grains comprising high levels of thioredoxin and / or thioredoxin reductase, comprising transforming the plants with an expression cassette as mentioned above;

A first plant comprising a heterologous expression cassette comprising a promoter regulated and regulated by a transcriptional activator operably linked to a DNA sequence encoding thioredoxin and / or thioredoxin reductase, to the transcriptional activator Pollination with a pollen from a second plant comprising a heterologous expression cassette comprising a promoter operably linked to a DNA sequence encoding a transcriptional activator capable of regulating a regulated promoter and

A method for producing a grain comprising high levels of thioredoxin and / or thioredoxin reductase, comprising recovering the grain from the pollinated plant as in the above step.

In addition, the present invention provides:

Use of plants or plant materials according to the invention as animal feed.

The invention described herein is applicable to all cereal crops, in particular corn, soybeans, wheat and barley, most particularly corn and soybeans, particularly corn. Expression in grains of transformed thioredoxin and / or thioredoxin reductases modifies the quality of the material (seed) to be introduced into the grain processing, modifies the quality of the material derived from the grain processing, and It is a means of minimizing the yield of seed components during processing (increasing efficiency), changing the processing method and creating new uses for seed-derived fractions or components from the grinding stream.

Wet grinding process

Wet grinding is the process of separating the starch, protein and oil components of cereals, most often grains such as corn. This process is distinguished here from the dry grinding process, which simply powders the grains. The first step in the wet grinding process is usually an immersion step in which grains are immersed in water under carefully controlled conditions to soften nuclei to facilitate separation of components. Since the oil-containing embryos float on the surface of the aqueous solution, they are removed and purified by separating the whole bulb from the protein by watering and dehydration, grinding, screening, centrifugation and washing. The main difficulty is to free the starch particles from the complex matrix of protein and cell wall material that make up the endocrine of the grains. One reason for this difficulty is the presence of intermolecular or intramolecular disulfide bonds that make the protein matrix less soluble and less sensitive to proteolytic enzymes and which prevent the release of starch particles from the protein matrix in cereals. Is considered. At present, the first means to reduce these bonds is to soak grains in the presence of sulfur dioxide, which is costly, environmentally unfriendly and not best available.

Certain mutations have a beneficial effect on the protein matrix of corn nucleus endosperm (powdery and opaque), but impair nuclear integrity. Transforming thioredoxin expression provides certain of these advantages without causing certain nuclear integrity problems associated with these mutations.

Post-harvest or process-dependent activity of thioredoxin has the same beneficial effect. For example, in certain embodiments, thioredoxin enzymes are targeted to and accumulated within the cell compartment. Protein reduction occurs following physical destruction of the seed. In another embodiment, the inactive endosperth thioredoxin is activated upon immersion. In a preferred embodiment, the present invention provides that the heat-transforming thioredoxin and thioredoxin reductases, such as thioredoxin and thioredoxin reductase, are sufficiently high, except at high temperatures (eg 50 ° C. or higher). Provided are plants that express thioredoxin and thioredoxin reductases derived from inactive superthermophilic organisms. In certain embodiments, thermally stable thioredoxin and thioredoxin reductase are synergized by starch through expression of other thermally stable enzymes in endocrine.

Feed use

In addition, expression of the transformed thioredoxin and / or thioredoxin reductase in cereals is particularly useful for improving grain properties associated with digestibility in animal feed. Feed protein susceptibility to proteases is a function of time and protein structure. Nuclear cracking, like steam flakes, is often used in feed formulations. Both of these processes are designed to contribute to nuclear digestibility. Preference is given to softer nuclei where integrity can be more readily destroyed on animals. Structural inhibition and crosslinking between proteins are major determinants of protease sensitivity. Modifying these bonds by enhanced thioredoxin expression promotes digestion.

Corn Dry Grinding Process

Protein content and quality are important determinants of flaking grit and martha production. Reduction of disulfide bonds alters the properties of corn flours, particularly flours made from high protein white corn varieties, suitable for use as flour substitutes.

Soybean crushing process

More than half of US soybean crops are crushed or ground, and protein quality in the resulting low fat soy flour or skim soy flour (or grit) is important for subsequent processing. Protein yield and quality from soybean process streams are economically important and highly dependent on protein structure. Enhancement of thioredoxin activity through expression of transformed thioredoxin and / or thioredoxin reductase enhances protein solubility in the water soluble protein fraction, thereby increasing yield. Aqueous extraction of degreased soy flour under basic conditions facilitates recovery. Enhancement of thioredoxin activity through the expression of transformed thioredoxin and / or thioredoxin reductase also lowers the pH required for efficient extraction, reducing calcium or sodium hydroxide influx, as well as subsequent acid precipitation. It reduces acid intake into the water, enables efficient recovery of protein without alkali damage, and reduces water consumption and treatment plant waste emissions (containing the actual biological oxygen demand load).

Protein redox status affects important functional properties provided by soy protein, such as solubility, water absorption, viscosity, stickiness / adhesion, gelling and elasticity. Fiber removal during the soy protein concentrate production process and soy protein digest hydrolysis by proteases can be enhanced by increasing thioredoxin activity as described herein. Similarly, as described for maize, the increase in thioredoxin activity through expression of transformed thioredoxin and / or thioredoxin reductase may be attributed to the functionality of enzyme-active soy flour and soybeans in animal feed. Promotes digestibility of minute fractions and steam flake fractions.

Although it is desirable for the transformed thioredoxin and / or thioredoxin reductase to be targeted and thermally stable to the cell compartment as described above, to avoid significant adverse effects on storage protein accumulation, during and during seed development provided. Modification of protein quality during may be faced as a result of thioredoxin activity during seed development. Alternatively, thioredoxin can be added as process enzyme because cleavage of disulfide bonds (as opposed to corn wet grinding) is not necessary until after grain integrity is broken (crushing and oil extraction).

Selection of thioredoxin and thioredoxin reductase for heterologous expression:

Thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI) genes are not only eukaryotic cells, true bacteria, but also superthermophilic organisms such as Methanococcus jannaschii and arcaeogle. It is found in hard-tolerant bacteria, including Robus pergidus. The choice of specific genes depends somewhat on the intended use. For the process of the invention, preferred thioredoxins have the following properties:

1. Thermal stability

Thioredoxin and related proteins from S. aureus have been found to have enhanced stability at high temperatures (> 50 ° C.) and relatively low activity at ambient temperatures. Expression of TRX and / or TR from hardy bacteria, such as Staphylococcus aureus, such as Metanococcus zannaschy and Arcaeoglobus pulgidus, is preferred for expression during seed development, so that grains are immersed at elevated temperature. Tioredoxin activity is not significantly enhanced until after treatment or treatment. Most grain processing methods include or are compatible with high temperature steps. Therefore, thermally stable thioredoxin and thioredoxin reductase are preferred. Thermal stability means that the enzyme is at a high temperature, for example at least 40 ° C., most preferably at least 50 ° C., for example 45 to 60 ° C., or even higher, for example 45 to 60 ° C. for a wet grinding process. Preferred active at 95 ° C.

2. Substrate Specificity

Undesirable effects on seed development can be reduced by the selection of thioredoxin, which acts preferentially on certain proteins such as structural proteins in the matrix and has low activity against essential metabolic enzymes. Various TRXs have been found to differ in reactivity with enzymes under redox control. Thus, it is possible to select TRX that can essentially act on the desired target while minimizing the undesirable side effects of overexpression.

Suitable thermally stable thioredoxin and thioredoxin reductases comprise the following sequence:

Sequence of thioredoxin from Metanococcus zanaceki (SEQ ID NO: 1 gi | 1591029)

MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPQKAMEYGIMAVPTIVINGDVEFIGAPTKEALVEAIKKRL

Sequence of thioredoxin from Arcaeoglobus pergidus (SEQ ID NO: 2: gi | 2649903) (trx-1)

MPMVRKAAFYAIAVISGVLAAVVGNALYHNFNSDLGAQAKIYFFYSDSCPHCREVKPYVEEFAKTHNLTWCNVAEMDANCSKIAQEFGIKYVPTLVIMDEEAHVFVGSDEVRTAIEGMK

Sequence of thioredoxin from Arcaeoglobus pulgidus (SEQ ID NO: 3 gi | 2649838) (trx-2)

MVFTSKYCPYCRAFEKVVERLMGELNGTVEFEVVDVDEKRELAEKYEVLMLPTLVLADGDEVLGGFMGFADYKTAREAILEQISAFLKPDYKN

Sequence of thioredoxin from Arcaeoglobus pulgidus (SEQ ID NO: 4 gi | 2649295) (trx-3)

MDELELIRQKKLKEMMQKMSGEEKARKVLDSPVKLNSSNFDETLKNNENVVVDFWAEWCMPCKMIAPVIEELAKEYAGKVVFGKLNTDENPTIAARYGISAIPTLIFFKKGKPVDQLVGAMPKSELKRWVQRNL

Sequence of thioredoxin from Arcaeoglobus pulgidus (SEQ ID NO: 5: gi | 2648389) (trx-4)

MERLNSERFREVIQSDKLVVVDFYADWCMPCRYISPILEKLSKEYNGEVEFYKLNVDENQDVAFEYGIASIPTVLFFRNGKVVGGFIGAMPESAVRAEIEKALGA

Sequence of thioredoxin reductase (trxB) from Metanococcus jannaskyi (SEQ ID NO: 6 gi | 1592167)

MIHDTIIIGAGPGGLTAGIYAMRGKLNALCIEKENAGGRIAEAGIVENYPGFEEIRGYELAEKFKNHAEKFKLPIIYDEVIKIETKERPFKVITKNSEYLTKTIVIATGTKPKKLGLNEDKFIGRGISYCTMCDAFFYLNKEVIVIGRDTPAIMSAINLKDIAKKVIVITDKSELKAAESIMLDKLKEANNVEIIYNAKPLEIVGEERAEGVKISVNGKEEIIKADGIFISLGHVPNTEFLKDSGIELDKKGFIKTDENCRTNIDGIYAVGDVRGGVMQVAKAVGDGCVAMANIIKYLQKL

Sequence of thioredoxin from Arcaeoglobus pulgidus (SEQ ID NO: 7: gi | 2649006) (trxB)

MYDVAIIGGGPAGLTAALYSARYGLKTVFFETVDPVSQLSLAAKIENYPGFEGSGMELLEKMKEQAVKAGAEWKLEKVERVERNGETFTVIAEGGEYEAKAIIVATGGKHKEAGIEGESAFIGRGVSYCATCDGNFFRGKKVIVYGSGKEAIEDAIYLHDIGCEVTIVSRTPSFRAEKALVEEVEKRGIPVHYSTTIRKIIGSGKVEKVVAYNREKKEEFEIEADGIFVAIGMRPATDVVAELGVERDSMGYIKVDKEQRTNVEGVFAAGDCCDNPLKQVVTACGDGAVAAYSAYKYLTS

Genes encoding such proteins for use in the present invention are preferably designed by reverse translation using preferred codons in plants, as described more fully below, to enhance G-C content and remove harmful sequences. The activity of the protein can be enhanced by DNA shuffling or other means as described below. Thus, the present invention includes such proteins, particularly proteins derived from substantially similar proteins that possess thioredoxin or thioredoxin reductase activity.

In order to manipulate thioredoxin expression in seeds for activity during grain development, promoters that direct seed specific expression of TRX and TR are preferred, which has the desired effect on storage proteins where enzymes may be desirable in certain applications. Target for storage so that it has a. However, in the present invention, it is generally desirable to manipulate thioredoxin and / or thioredoxin reductase expression in seeds for accumulation and inactivation during grain development. To generate seeds that express transformed thioredoxin and / or thioredoxin reductase with little effect on normal seed development, for example, several measures are used:

(i) Active thioredoxin or thioredoxin reductases are compartmentalized such that they are targeted to or expressed in the white body, for example, so that they rarely interact with the target protein. The pigment targeting sequence is used for direct accumulation in the white body. Alternatively, thioredoxin and / or thioredoxin reductase is targeted for extracellular location in the cell wall using secretion signals. Or lastly, in the case of monocotyledonous plants, during seed development, expression in cell types such as whistle is used to isolate thioredoxin and / or thioredoxin reductase from the storage components of the remaining endoderm.

(ii) engineer expression of thioredoxin and / or thioredoxin reductases derived from thermophilic organisms. Enzymes that have little or no activity at ambient temperature (as high as 38-39 ° C. at the site) will probably cause little problems during development. Thus, preferably, the enzyme is primarily at a high temperature, for example at least 40 ° C., most preferably at 45 to 60 ° C., or even higher, for example 45 to 95 ° C. for wet grinding processes. Active.

(iii) transcriptional activation activated in minutes by plants expressing thioredoxin and / or thioredoxin reductases, e.g., chemically inducible promoters, wound inducible promoters, or transcriptional activators Placed under the control of a zero regulated promoter.

(iv) tees with specific requirements for specific thioredoxin reductases such that the activity of thioredoxin or thioredoxin reductase is appropriately controlled by the availability of each suitable thioredoxin reductase or thioredoxin. Use oredoxin. For example, thioredoxin and thioredoxin reductase are expressed in different plants so that the active combination is only available in the seed in minutes by the plant expressing the complement enzyme. Alternatively, thioredoxin or thioredoxin reductase is sequestered in cells, for example, pigments, vacuoles or apoplasts, such that grains are not available until processed.

Grain processing method

Accordingly, the present invention provides a novel method of enhancing the separation of starch from a protein matrix using thioredoxin and / or thioredoxin reductase. In a first embodiment, the thioredoxin activity is various, including wet grinding, dry grinding, oilseed processing, soybean processing, wheat processing and flour / dough quality, most particularly wet grinding of grains, especially corn. It has been found to be useful in seed treatment applications.

Thus, the present invention comprises dipping grain in the presence of supplementary thioredoxin and / or thioredoxin reductase, and separating the starch and protein components of the grain,

Improve grinding efficiency or improve grinding yield,

Enhance the separation efficiency of starch and protein,

Improve the yield of starch and soluble protein from cereals,

Provide methods for enhancing protein solubility in water or other solvents.

Typically, the dipping process occurs before the grinding process, but may occur afterwards, and one or more grinding or dipping steps can be present in the extraction process to enhance protein yield from the seed during or after dipping. . Preferably, supplemental thioredoxin and / or thioredoxin reductase is provided by the expression of the transgene in the plant from which the crop will be harvested.

In addition, the present invention

The use of thioredoxin or thioredoxin reductase in a method for improving the grinding efficiency or enhancing the grinding yield of starch or protein, for example in one of the methods described above,

Immersion water containing an amount of thioredoxin and / or thioredoxin reductase effective to facilitate separation of the starch from the protein in the grain,

Cereals exposed to thioredoxin in an amount effective to promote separation from the starch protein and

Provides starch or protein prepared by the method described above.

The activity of thioredoxin in the above methods can be enhanced by supplementing the immersion water with thioredoxin reductase and / or NADPH. Other components typically present in wet grinding immersion water may also be present, such as bacteria producing lactic acid. Preferably, the dipping process is carried out for 22 to 50 hours at a temperature of about 52 ℃, it is preferred that thioredoxin is stable under the above conditions.

Cereals may be oilseed seeds, for example soybean, sunflower or rapeseed, preferably soybean seeds, such as soybeans, or grain seeds, for example corn, wheat, oats, barley, rye or rice, most preferably Monocot seeds, such as corn.

Thioredoxin can be any protein having a thiol group that can be reversibly oxidized by NADPH in the presence of thioredoxin reductase to form disulfide bonds and be reduced. Preferably, thioredoxin is derived from thermophilic organisms as described above. Thioredoxin and / or thioredoxin reductases for use in the present invention may be engineered into engineered microorganisms, such as yeast or Aspergillus, or, for example, high pi alpha-amylase promoters or other gibberellin- Properly produced in engineered plants that are highly highly expressive in barley, for example, under the control of a promoter that is active during malting, such as a dependent promoter. Thioredoxin (in the secreted or extracted form or combination with the producer organism or part thereof) is then added to the immersion water.

As an alternative or supplement to the addition of thioredoxin to immersion water, the enzyme can be expressed directly in the seed to be milled. Preferably, the enzyme is expressed during grain maturation or during the regulatory process.

Thus, in a further embodiment, the present invention

Preparation of thioredoxin on an industrial scale in transgenic organisms, for example in cereals or microorganisms such as barley or corn, for example yeast or aspergillus, for example A method comprising culturing a transgene carrying a chimeric gene expressing thioredoxin and optionally isolating or extracting thioredoxin,

So that the quality of the protein in the seed is affected by thioredoxin, which is endogenously synthesized during seed development or during the soaking process, so as to exclude or reduce the need for exogenous chemicals or enzymes to control before grinding. Methods of using transgenic plants that produce enhanced quality of thioredoxin during maturation or germination,

The quality of the protein during seed maturation or germination so that the quality of the protein in the seed is influenced by thioredoxin during seed development or during the soaking process, thereby eliminating or reducing the need for exogenous chemical or enzyme control prior to the grinding process. A method of making a transgenic plant that produces an enhanced quality of oredoxin and

A method of grinding grains that further enhances starch and soluble protein yields using transformed seeds containing thioredoxin.

Expression of Thioredoxin and Thioredoxin Reductase in Transgenic Organisms

The invention also encompasses transgenic organisms carrying a chimeric expression cassette in the genome comprising a coding region encoding a thermally stable thioredoxin or thioredoxin reductase under operative control of a promoter.

Preferably, the transgenic organism expresses thioredoxin and / or thioredoxin reductase in a form not naturally occurring in the plant of the species, or the thioredoxin is naturally occurring in the plant of the species. It is a plant that expresses higher level than it does.

Preferably, thioredoxin is expressed in the seed during seed development, and is therefore preferably placed under the control of a seed specific promoter. Optionally, expression of thioredoxin is placed under the control of a promoter that is inducible or regulated by a transcriptional activator and, if necessary, is activated by chemical induction or hybridization with the transcriptional activator. Thioredoxin is suitably targeted to the vacuoles of the plant by fusion with vacuole targeting sequences.

In the present invention, a thioredoxin coding sequence is fused to a promoter active in a plant and transformed into the nuclear genome or the chromatin genome. The promoter is preferably a seed specific promoter, for example a gamma-zein promoter. Alternatively, the promoter may be a chemically inducible promoter, for example a tobacco PR-1a promoter, or a promoter whose transcription activator is regulated by a chemically induced transcription activator present under the control of a chemically induced promoter. In certain circumstances, a configuration promoter may be used, such as a CaMV 35S or Gelvin promoter. Chemically inducible promoters can be used to activate expression of the thioredoxin transformed into the plant at the appropriate time by application of the leaves of the chemical inducer.

Alternatively, the thioredoxin coding sequence is placed under the control of a promoter regulated with a transcriptional activator, and the plant transformed with the sequence is crossed with a second plant expressing the transcriptional activator to achieve expression. In a preferred form of the method, the first plant containing the thioredoxin coding sequence is seed parent and male infertility, while the second plant expressing a transcriptional activator is water. Expression in the seed of thioredoxin may be, for example, a promoter regulated by a transcriptional activator with a transcriptional activator expressed by the transcriptional activator gene from the first plant and pollinated by the second plant. Achieved by corrosion of the first plant and the second plant such that thioredoxin is expressed in the seed of the first plant.

Accordingly, the present invention relates to a plant expressing thioredoxin and / or thioredoxin reductase, eg, thioredoxin and / or thioredoxin reductase, which is not naturally expressed in a plant, eg, a nucleus or The heterologous DNA sequence encoding thioredoxin, which is stably integrated in the chromatin DNA, is preferably activated or under the control of a chemically inducible promoter operably linked to an inducible promoter, eg, an inducible promoter. Provided are plants that express under the control of a zero regulated promoter, wherein the corresponding transcriptional activator is placed under inducible promoter control or expressed in the second plant such that the promoter is activated by hybridization with the second plant, Thioredoxin or thioredoxin reductase is preferably thermally stable and the plant also Seeds optionally treated (eg undercoat or sheathed) and / or packaged, for example seeds placed in bags containing instructions for use and seeds harvested therefrom for use in the grinding process as described above. Induce.

The transgenic plant of the invention may optionally further comprise a gene for enhanced production of thioredoxin reductase and / or NADPH.

In addition, the present invention

A process for the preparation of thioredoxin, comprising growing a thioredoxin-expressing plant as described above,

A process for preparing starch and / or protein, comprising extracting starch or protein from seeds harvested from plants as described above;

We further provide a wet grinding method comprising soaking seeds from thioredoxin-expressing plants as described above and extracting starch and / or protein therefrom.

In addition, the present invention

Thioredoxin or thioredoxin reductases, preferably thermophilic organisms, eg suffering bacteria, eg M as described above. Jannasuki or A. An expression cassette expressible in a plant comprising a coding region for thioredoxin derived from Pergidus, wherein the coding region is optimized to contain the desired codons in the plant, and the coding region is a promoter and terminator that acts in the plant. Operably linked to a sequence, the promoter is preferably a seed specific promoter or an inducible promoter, for example a promoter that is chemically inducible or regulated with a transcriptional activator, for example a pigment or nuclear expressable The expression cassette is a promoter, for example a promoter that is regulated by a transcriptional activator regulated by a nuclear transcriptional activator (e.g., when the transcriptional activator is a T7 RNA polymerase, expression is present under the control of an optionally inducible promoter T7. Is a promoter),

A vector comprising an expression cassette expressible in said plant,

Plants transformed with the vector or

Further providing a transgenic plant comprising an expression cassette expressible in said plant in a genome, eg, a nuclear genome or a chromosomal genome.

In addition, the present invention provides a sterile or male sterile agent comprising a heterologous expression cassette comprising a promoter regulated and regulated with a transcriptional activator operably linked to a DNA sequence encoding thioredoxin or thioredoxin reductase. 1 plant is pollinated with pollen from a second plant comprising a heterologous expression cassette comprising a promoter operably linked to a DNA sequence encoding a transcriptional activator capable of regulating a promoter regulated by the transcriptional activator, A process for producing grains comprising high levels of thioredoxin or thioredoxin reductase, including recovering from plants so polluted from cereals.

Justice

To ensure clear and consistent understanding of the specification and claims, the following definitions are provided:

As used herein, " expression cassette " means a DNA sequence capable of directing expression of a gene in plant cells, including a promoter operably linked to a desired coding region, operably linked to an end region. do. The coding region usually encodes a protein of interest, but encodes antisense RNA or non-translated RNA that inhibits the expression of a specific gene, eg, antisense RNA, in the direction of the desired functional RNA, eg, sense or antisense. You may. The gene may be a chimeric, meaning that one or more components of the gene are heterologous to one or more other components of the gene. In addition, genes may be genes that occur naturally but are obtained in recombinant form useful for gene transformation of plants. Typically, however, the expression cassette is heterologous to the host, ie the DNA sequence specific to the expression cassette does not naturally occur in the host cell and must be introduced into the host cell or ancestor of the host cell.

A " nuclear expression cassette " is an expression cassette that is integrated into the nuclear DNA of a host.

A " chromosomal expression cassette " is an expression cassette that is integrated into the pigment DNA of a host. The pigment expression cassettes described herein, for example, encode protein of antisense mRNA that inhibits the expression of two or more cistron coding sequences of interest, wherein one of the coding sequences is clpP or other pigment protease, Polycistron operon, which contains the other coding sequence or enhances the accumulation of the desired sequence) under the control of a single promoter, eg, a promoter controlled by a transcriptional activator.

As used herein, “ heterologous ” means “different natural origin”. For example, when a plant is transformed with a gene derived from another organism, in particular another species, the gene is heterologous to the offspring of the plant and the plant carrying the gene.

" Homochromosome " means a plant, plant tissue or plant cell in which the entire plastid is genetically identical. This is normal in plants when the plastids are not transformed, mutated or otherwise genetically modified. In different tissues or developmental phases, the plastids can take different forms, such as chloroplasts, primitives, ethioplasts, whites, variegates and the like.

An " inducible promoter " is a promoter that initiates transcription only when a plant is exposed to some unique external stimulus, which is distinguished from a constitutive promoter or a promoter specific to a specific tissue or organ or developmental organ. Particularly preferred inducible promoters for the present invention are promoters which are chemically inducible or regulated with transcriptional activators. Chemically inducible promoters may be plant-derived promoters, such as those in the systemic acquisition resistance pathways, such as PR promoters such as PR-1, PR-2, PR-3, PR-4 and PR-5 promoters. Including the tobacco PR-1a promoter and the Arabidopsis PR-1 promoter, which initiate transcription, particularly when plants are exposed to chemicals associated with BTH. See US Pat. No. 5,614,395, incorporated herein by reference. And WO 98/03536. In addition, chemically inducible promoters may be used in receptor-mediated systems such as steroid-dependent gene expression, copper-dependent gene expression, tetracycline-dependent gene expression, in particular the glazes described in PCT / EP 96/04224. Expression systems using USP receptors from Drosophila mediated by GI growth hormones and agonists thereof, as well as receptors as described in PCT / EP 96/00686, which is incorporated herein by reference. And those derived from other organisms, such as expression systems using combinations. The chemically inducible promoter may be directly linked to the thioredoxin gene, or the thioredoxin gene may be under the control of a promoter regulated by a transcriptional activator, while the gene for the transcriptional activator may be a chemically inducible promoter [Overview, C. Gatz, "Chemical Control of Gene Expression", Annu. Rev. Plant Physiol. Plant Mol. (1997) 48 : 89-108. The promoter regulated by a transcriptional activator is more fully described below, which may be induced by hydriding a plant comprising the thioredoxin gene under the control of a promoter regulated by a transcriptional activator with a second plant expressing the transcriptional activator. have.

An " isolated DNA molecule " is a nucleotide sequence that is not a natural product, which is separated from its natural environment by a human hand. The isolated nucleotide sequence may be present in purified form or may be present in an unnatural environment such as, for example, a transforming host cell.

A " protein " as defined herein is the entire protein encoded by the corresponding nucleotide sequence or the portion of a protein encoded by the corresponding portion of the nucleotide sequence.

An " isolated protein " is a protein that is not a natural product, encoded by an isolated nucleotide sequence. An isolated protein may be present in purified form, or may be present in an unnatural environment, such as a transgenic host cell that does not normally express the protein or that may be expressed in different forms or in different amounts within an untransformed host cell of the homologous gene. Can be.

" Plant " means any plant or part of a plant in the growing season and is specifically intended to include damaged, collapsed or dead plants and plant materials, as well as viable plants, inserts, cell or tissue cultures and seeds. .

" DNA shuffling " is a method of introducing mutations or translocations into a DNA molecule, preferably randomly, or exchanging DNA sequences, preferably between two or more DNA molecules, randomly. DNA molecules generated from DNA shuffling are shuffled DNA molecules, which are non-natural DNA molecules derived from one or more template DNA molecules. The shuffled DNA encodes a modified enzyme with respect to the enzyme encoded by the template DNA and preferably has altered biological activity with respect to the enzyme encoded by the template DNA.

In the broadest sense, the term “ substantially similar ”, when used herein in reference to a nucleotide, refers to a reference nucleotide sequence in which a corresponding change occurs in a corresponding amino acid, for example, that does not affect polypeptide function. By nucleotide sequence corresponding to a reference nucleotide sequence encoding a polypeptide having a structure and function substantially the same as the polypeptide encoded by. Preferably, substantially similar nucleotide sequences encode polypeptides encoded by a reference nucleotide sequence. The percentage of homology between substantially similar nucleotide sequences and reference nucleotides is preferably at least 80%, more preferably at least 85%, preferentially at least 90%, more preferably at least 95%, even more preferentially 99 More than% Sequence comparisons are described in Smith-Waterman sequence alignment algorithms [see, for example, Waterman, MS Introduction to Computational Biology: Maps, sequences and genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391-0 or http://www-hto.usc.edu/software/seqaln/index.html ]. Use the LocalS program, version 1.16, with the following parameters: Acquisition: 1, Mismatch Penalty: 0.33, Open-Gap Penalty: 2, Extended-Gap Penalty: 2.

The nucleotide sequence "substantially similar" to the reference nucleotide sequence is 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA washed with 2X SSC at 50 ° C, 0.1% SDS at 50 ° C, more preferably 7 % Sodium Dodecyl Sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA washed with 1X SSC at 50 ° C., 0.1% SDS at 50 ° C., more preferably 7% Sodium Dodecyl Sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA washed with 0.5X SSC at 50 ° C, 0.1% SDS at 50 ° C, preferentially 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 0.1X SSC at 50 ° C, 0.1 at 50 ° C Reference nucleotide sequence in 1 mM EDTA washed with% SDS, more preferentially 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 0.1 X SSC at 50 ° C., 1 mM EDTA washed with 0.1% SDS at 65 ° C. Hybridize to

The term “ substantially similar ” when used in reference to a protein refers to a reference having a structure and function substantially the same as that of the reference protein, where the only change in amino acid occurs that does not affect, for example, polypeptide function. It means a protein corresponding to a protein. When used for a protein or amino acid sequence, the percentage of homology between substantially similar protein or amino acid sequences and the reference protein or amino acid sequence is preferably at least 80%, more preferably at least 85%, preferentially at least 90%, More preferably at least 95% and even more preferably at least 99%.

A " transcriptional activator " is a protein that can be combined with the protein itself or with one or more additional proteins to cause transcription of the coding region under the control of a promoter regulated with the corresponding transcriptional activator. Examples of transcriptional activator systems include the phage T7 gene 10 promoter whose transcriptional activation is dependent on specific RNA polymerases such as phage T7 RNA polymerase. Transcriptional activators are typically RNA polymerases that can initiate transcription by directly activating a promoter or interacting with a specific promoter, for example by inhibiting the expression or accumulation of the repressor protein to inactivate the repressor. Or DNA binding proteins. The DNA binding protein may be a chimeric protein comprising a binding region (eg, a GAL4 binding region) that is linked to a suitable transcriptional activator region. Certain transcriptional activator systems may possess multiple transcriptional activators, eg, polymerases, as well as promoters that require subunits (sigma factors) specific for promoter recognition, DNA binding, or transcriptional activation. Transcription activators are preferably heterologous to the plant.

Modification of Microbial Genes to Optimize Nuclear Expression in Plants

If desired, the cloned thioredoxin gene described herein is modified for expression in a transgenic plant host. For example, it may be necessary to modify the gene in order to achieve transgenic expression in plants of genes derived from microbial origin and to achieve their expression in plants. In particular, bacterial ORFs encoding individual enzymes but encoded by the same transcript in natural microorganisms are best expressed in plants for individual transcripts. To accomplish this, each microbial ORF is isolated separately and cloned in a cassette providing a plant promoter sequence at the 5 'end of the ORF and a plant transcription terminator at the 3' end of the ORF. An isolated ORF sequence preferably comprises an initiating ATG codon and an ending STOP codon, but may comprise additional sequences other than the initiating ATG codon and the ending STOP codon. In addition, an ORF can be cleaved, but a truncated version that still retains the necessary activity, particularly in the case of long ORFs, is preferred for expression in the transgenic organism.

" Plant promoter " and " plant transcription terminator " refer to promoters and transcription terminators that operate in plant cells. This includes promoters and transcriptional terminators that may be derived from non-plant origin, such as viruses (eg, Cauliflower Mosaic Virus or Agrobacterium Ti or Ri plasmid genes of the Opin Synthase). Promoters and terminators).

In certain cases, modifications to sequences adjacent to the ORF coding sequence may not be required, but in this case it is sufficient to isolate the fragment containing the desired ORF and insert it downstream of the plant promoter. However, preferably, adjacent microbial sequences attached upstream of the ATG and downstream of the STOP codon should be minimized or eliminated. In practice, this construction may vary depending on the availability of the restriction site.

In other cases, expression of genes derived from microbial origin can provide a problem in expression. These problems are well characterized in the art, and are particularly common for genes derived from specific origins such as Bacillus. Modifications of such genes can be undertaken using techniques well known in the art. The following problems are typical of the problems you may encounter:

1. Codon Usage

Preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Codon usage in cloned microbial ORFs and comparison of plant genes (and in particular genes from target plants) will allow identification of codons in ORFs that should preferably be changed. Typically, plant evolution tends to strongly select nucleotides C and G at the third base position of the monocotyledonous plant, whereas dicotyledonous plants often use nucleotides A or T at these positions. By modifying the genes to mix the desired codon usage for specific target transgenic species, many of the problems described below for GC / AT content and inadequate splicing will be overcome.

2. GC / AT content

The GC content of plant genes is typically at least 35%. ORF sequences rich in A and T nucleotides can cause some problems in plants. First, the motif of ATTTA is considered to cause destabilization of the message and is found at the 3 'end of many RNAs. Second, generation of polyadenylation signals such as AATAAA at inappropriate locations in the message is considered to cause immature cleavage of transcription. In addition, monocotyledonous plants will recognize AT-rich sequences as splice sites (see below).

3. Sequences Adjacent to Initiating Methionine

Plants differ from microorganisms in that their messages do not have defined ribosomal binding sites. Rather, ribosomes are considered scanning for the first available ATG attached to the 5 'end of the message to initiate decryption. Nevertheless, selectivity exists for certain nucleotides adjacent to ATG, and expression of microbial genes may be enhanced by inclusion of eukaryotic consensus translational initiators in ATG. Clontech (1993/1994 catalog, page 210) has been developed for plant-derived bacteria. The sequence GTCGACC ATG GTC (SEQ ID NO: 8) was proposed as a consensus translational initiator for the expression of the E. coli uidA gene. In addition, Josh (NAR 15 : 6643-6653 (1987)) compared a number of plant sequences adjacent to ATG and proposed the consensus TAAACA ATG GCT (SEQ ID NO: 9). In situations where expression of the microbial ORF is difficult in plants, translation can be enhanced when one of the sequences is included in the starting ATG. In such cases, the last three nucleotides in the consensus may not be suitable for inclusion in the modified sequence due to modification of the second AA residue. Preferred sequences adjacent to the initiation methionine may differ between different plant species. Investigation of the 14 maize genes in the GenBank database gives the following results:

Position before initiation ATG in 14 maize genes

-10 -9 -8 -7 -6 -5 -4 -3 -2 -One

C 3 8 4 6 2 5 6 0 10 7

T 3 0 3 4 3 2 1 1 1 0

A 2 3 1 4 3 2 3 7 2 3

G 6 3 6 0 6 5 4 6 1 5

Such assays can be performed on the desired plant species incorporating the thioredoxin or thioredoxin reductase gene and modifying the sequence adjacent to the ATG to introduce the desired nucleotides.

4. Removal of nonconforming splice sites

Genes that are cloned from non-plant origin and not optimized for expression in the plant may also contain motifs that can be recognized and cleaved in the plant as 5 'or 3' splice sites, resulting in a truncated and deleted message. . These sites can be removed using the methods described in patent application WO 97/02352, which is incorporated herein by reference.

Methods for modification of sequences adjacent to coding sequences are well known in the art. If the initial expression of the microbial ORF is low and it is deemed suitable for modification to the sequence as described above, the construction of the synthetic gene can be accomplished according to methods well known in the art. This is described, for example, in published patent publications EP 0 385 962, EP 0 359 472 and WO 93/07278. In most cases, it is desirable to assay expression of gene constructs using transient assay protocols (well known in the art) prior to delivery to transgenic plants.

The main advantage of chromatin transformation is that the pigment can generally express bacterial genes without substantial modification. Application of codons as described above is not required and the chromosomes may express multiple open reading frames under the control of a single promoter.

Construction of Plant Transformation Vectors and Selectable Markers

Many transformation vectors are available for plant transformation, and the genes of the invention can be used with any of these vectors. The choice of vector for use will depend on the desired transformation technique and the target species for transformation. For certain target species, different antibody or herbicide selection markers may be preferred. Selection markers commonly used in transformation include nptII genes and related antibodies that confer resistance to kanamycin (Vieira & Messing, Gene 19 : 259-268 (1982); Bevan et al., Nature 304 : 184-187 (1983)], bar genes that provide resistance to the herbicide phoSph I notrycin (White e tal., Nucl Acids Res 18 : 1062 (1990), Spencer et al. Theor Appl Genet 79 : 625-631 (1990)], hpt gene that provides resistance to antibody hygromycin [Blochinger & Diggelmann, Mol Cell Biol 4 : 2929-2931], dhfr which provides resistance to methotrexate Mannose-6-phosphate, which provides the ability to metabolize mannose, described in the gene (Bourouis et al., EMBO J. 2 (7) : 1099-1104 (1983)) and US Pat. No. 5,767,378. Isomerase gene.

Requirements for the Construction of Plant Expression Cassettes

The gene sequence to be expressed in the transgenic plant is first combined in an expression cassette upstream of a suitable promoter and upstream of a suitable transcription terminator. These expression cassettes can then be readily delivered to the plant transformation vectors described above.

1. Select promoter

The choice of promoter used in the expression cassette will determine the spatial and temporal expression patterns of the transgene in the transgenic plant. The selected promoter may be used to express the transgene in a specific cell type (eg leaf epidermal cells, lobe cells, root cortical cells) or in specific tissues or organs (eg roots, seeds, leaves or flowers). This choice will indicate the desired thioredoxin biosynthesis site. In the present invention, seed specific promoters are preferred. Alternatively, the selected promoter can drive expression of the gene under a light-induced or other temporarily regulated promoter. A further alternative is to induce selected promoters by external stimulation, for example by application of specific chemical inducers or by hybridization with a second plant line that offers the possibility of inducing thioredoxin transcription only if necessary. Can be.

2. Warrior Terminator

Various transcription terminators are useful for use in expression cassettes. This leads to transcription termination and correct polyadenylation behind the transgene. Suitable transcriptional terminators and functions known in plants include CaMV 35S terminators, tml terminators, nopalin synthase terminators, and pea rbcS E9 terminators. They can be used in both monocotyledonous and dicotyledonous plants.

3. Sequences for Enhancing or Controlling Expression

A number of sequences have been found to enhance gene expression from within the transcription unit, and these sequences can be used with the genes of the invention to enhance expression of the transgenic plant.

Various intron sequences have been shown to enhance expression, particularly in monocotyledonous plant cells. For example, introns of the maize Adh1 gene have been found to significantly enhance the expression of wild-type genes under their homologous promoter when introduced into maize cells. Intron 1 has been shown to be particularly effective and to enhance expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop 1 : 1183-1200 (1987)). Within the same experimental system, introns from the corn bronze1 gene have a similar effect in enhancing expression. Intron sequences are typically introduced into plant transformation vectors, typically in a non-translated leader.

Many non-ready leader sequences derived from viruses are also known to enhance expression, which are particularly effective in dicotyledonous plant cells. Specifically, from leader sequences from Tobacco Mosaic Virus (TMV, "Ω-sequence"), Maize Chlorotic Mottle Virus (MCMV) and Alfalfa Mosaic Virus (AMV). Leader sequences have been shown to be effective for enhancing expression. See, eg, Gallie et al. Nucl. Acids Res. 15 : 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15 : 65-79 (1990).

4. Targeting Gene Products in Cells

Various mechanisms for targeting gene products are known to exist in plants, and the sequences controlling the functionalization of these mechanisms have been characterized in detail. The amino terminal sequence results in targeting for extracellular secretion from ER, apoplasts, and eukaryotic cells. Koehleer & Ho, Plant Cell 2 : 769-783 (1990). In addition, the amino-terminal sequence together with the carboxy-terminal sequence results in vacuole targeting of the gene product. Shinshi et al. Plant Molec. Biol. 14 : 357-368 (1990)]. By fusion of the above suitable targeting sequence to the desired transgene sequence, it is possible to direct the transformation product to the organelle or cell compartment. The signal sequence selected must comprise a known cleavage site and the constructed fusion must take any amino acid after the cleavage site necessary for cleavage. In certain cases, this requirement can be met by adding a few amino acids between the cleavage site and the transgene ATG or by substituting some amino acids with the transgene sequence.

The above-described mechanisms for the targeting of cells are used in conjunction with their homologous promoters as well as heterologous promoters to target specific cell targeting targets under the transcriptional control of promoters with expression patterns different from those of the promoter from which the targeting signal is derived. Can be achieved.

Examples of Expression Cassette Construction

The present invention includes expression of the thioredoxin gene under the control of any promoter that is expressible in a plant, regardless of the origin of the promoter.

The present invention also encompasses the use of any plant-expressable promoter with additional sequences necessary or selected for the expression of thioredoxin or thioredoxin reductase genes. Such sequences target transcriptional terminators, exogenous sequences that enhance expression (eg, introns (eg, Adh intron 1), viral sequences (eg, TMV-Ω)), and gene products to specific organelles and cell compartments. Include but are not limited to the sequence to be made.

Various chemical regulators may be used to induce the expression of thioredoxin or thioredoxin reductase coding sequences in plants transformed according to the present invention. In the context of the present specification, a "chemical regulator" includes a chemical known as an inducer to the PR-1a promoter in plants (see US Pat. No. 5,614,395) or a close derivative thereof. Preferred groups of chemically inducible modulators of the present invention are based on the benzo-1,2,3-thiadiazole (BTH) structure and are of the following kinds of compounds: benzo-1,2,3-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-thiadiazole-7-thiocarboxylic acid, 7-cyanobenzo-1,2 , 3-thiadiazole, benzo-1,2,3-thiadiazole-7-carboxylic acid amide, benzo-1,2,3-thiadiazole-7-carboxylic acid hydrazide, having 1 to 6 alkyl groups Benzo-1,2,3-thiadiazole carboxylate containing 2 carbon atoms, methyl benzo-1,2,3-thiadiazole-7-carboxylate, n-propyl benzo-1,2,3-thia Diazol-7-carboxylate, benzyl benzo-1,2,3-thiadiazole-7-carboxylate, benzo-1,2,3-thiadia 7-carboxylic acid sec-one include suitable derivatives thereof with butyl hydro-azide, and the like. Other chemical inducers may include, for example, benzoic acid, salicylic acid (SA), polyacrylic acid and their derivatives substituted, and suitable substituents include lower alkyl, lower alkoxy, lower alkylthio and halogen. Another group of modulators for the chemically inducible DNA sequences of the present invention are based on pyridine carboxylic acid structures, such as isoninicotinic acid structures, preferably haloisonicotinic acid structures. Preference is given to dichloroisonicotinic acid and its derivatives, for example lower alkyl esters. Suitable modulators of this class of compounds are, for example, 2,6-dichloroisonicotinic acid (INA) and lower alkyl esters thereof, in particular methyl esters.

Constitutive expression: actin promoter

Several isoforms of actin are known to express in most cell types, with the result that the actin promoter is a good choice for the constitutive promoter. In particular, promoters from the rice Act1 gene have been cloned and characterized. McElroy et al. Plant Cell 2 : 163-171 (1990)]. The 1.3 kb fragment of the promoter was found to contain all the regulatory factors required for expression in rice protoplasts. In addition, many expression vectors based on the Act1 promoter have been specifically constructed for use in monocotyledonous plants. McElroy et al. Mol. Gen. Genet. 231 : 150-160 (1991)]. It introduces Act1-intron 1, Adh1 5 'flanking sequences and sequences from Adh1-intron 1 (corn alcohol dehydrogenase gene origin) and CaMV 35S promoters. The vector with the highest expression is a fusion of 35S with Act1 intron or Act1 5 'flanking sequence and Act intron. Optimization of the sequence around the starting ATG (of the GUS reporter gene) also enhances expression. See McElroy et al. Mol. Gen. Genet. 231 : 150-160 (1991) can be readily modified for expression of the genes of the invention and are particularly suitable for use in monocotyledonous plant hosts. For example, promoter containing fragments can be removed from McElroy constructs and used to replace the dual 35S promoter in pCGN1761ENX, which is available for insertion or specific genes. The fusion gene so constructed can then be delivered to a suitable transformation vector. In a separate report, the rice Act1 promoter containing the first intron was also found to direct high expression in cultured barley cells. Chibbar et al. Plant Cell Rep. 12 : 506-509 (1993).

Constitutive expression: ubiquitin promoter

Ubiquitin is another gene product known to accumulate in many cell types, and its promoter has been cloned from several species for use in transgenic plants. See, for example, sunflower-Binet et al. Plant Science 79 : 87-94 (1991), corn-Christensen et al. Plant Molec. Biol. 12 : 619-632 (1989). Corn ubiquitin promoters occur in transgenic monocot plant systems and their sequences and vectors constructed for monocot plant transformation are described in patent publication EP 0 342 926. In addition, a vector (pAHC25) comprising its highly active in cell suspensions of a plurality of monocotyledonous plants when introduced by the corn ubiquitin promoter and the first intron and microblast bombardment is described by Taylor et al. Plant Cell Rep. 12: 491-495 (1993). Ubiquitin promoters are suitable for the expression of thioredoxin in transgenic plants, especially monocotyledonous plants. Suitable vectors are any of the transformation vectors described herein, modified by the introduction of a derivative of pAHC25 or a suitable ubiquitin promoter and / or intron sequence.

Root Specific Expression

Another preferred pattern of expression for thioredoxin and thioredoxin reductases of the present invention is root expression that enhances the extraction of starch and sugars from root crops such as, for example, sugar beet and potatoes. Suitable root promoters are described in de Framond, FEBS 290 : 103-106 (1991) or published patent application EP 0 452 269. This promoter is transferred to a vector suitable for insertion of the thioredoxin or thioredoxin reductase gene, eg, pCGN1716ENX, and subsequently the entire promoter-gene-terminator cassette is transferred to the desired transformation vector.

Wound-inducible promoter

Wound-inducible promoters may also be suitable for expression of the thioredoxin gene that is activated at harvest. Many such promoters are described in Xu et al. Plant Molec. Biol. 22 : 573-588 (1993), Logemann et al. Plant Cell 1 : 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22 : 783-792 (1993), Firek et al. Plant Molec. Biol. 22 : 129-142 (1993), Warner et al. Plant J. 3 : 191-201 (1993), all of which are suitable for use in the present invention. Logemann et al. Describe the 5 'upstream sequence of the wun1 gene of a dicotyledonous potato. Xu et al suggest that a wound-inducible promoter from the dicotyledonous plant, pin2, is active in rice, the monocotyledonous plant. In addition, Lormeier and Lehle describe the cloning of maize Wip1 cDNA that can be wound induced and used to isolate homologous promoters using standard methods. Similarly, Firek et al. Warner et al describe wound-derived genes from Asparagus officinalis, a monocotyledonous plant expressed at local wounds and pathogen invasion sites. . Using a cloning technique well known in the art, the promoter can be transferred to a suitable vector and fused to the thioredoxin gene of the present invention to express the gene at the wound site of the plant.

Expression Using Chromosome Targeting

See Chen & Jagendorf, J. Biol. Chem. 268 : 2363-2367 (1993) describe the successful use of chloroplast transgenic peptides for the introduction of heterologous transgenes. The peptide used was a transgenic peptide from the rbcS gene from Nicotiana plumbaginifolia [Poulsen et al. Mol. Gen. Genet. 205 : 193-200 (1986). Using the restriction enzymes DraI and SphI, or Tso509I and SphI, the DNA sequence encoding the transit peptide is cleaved from plasmid prbS-8B and manipulated for use in any of the above constructions. The DraI-SphI fragment extends from -58, corresponding to the starting rbcS ATG, to the site containing the first amino acid (also methionine) of the mature peptide immediately after the inlet cleavage site, while the Tsp509I-SphI fragment corresponds to the starting rbcS ATG Extends from -8 to the site comprising the first amino acid of the mature peptide. Thus, these fragments are thioredoxin or thioredoxin downstream of the exact fusion of the transient peptide, creating a transcriptional fusion with the non-ready leader of the selected promoter (eg 35S, PR-1a, actin, ubiquitin, etc.). It can be suitably inserted into the polylinker of any selected expression cassette that allows insertion of reductase.

Transformation of Dicotyledonous Plants

Transformation techniques of dicotyledonous plants are well known in the art and include techniques based on Agrobacterium and techniques that do not require Agrobacterium. Non-Agrobacterium techniques include direct uptake of exogenous genetic material by protoplasts or cells. This can be accomplished by PEG or electroporation mediated absorption, particle impact-mediated delivery or microinjection. Examples of these techniques are described in Paszkowski et al., EMBO J 3 : 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199 : 169-177 (1985), Reich et al., Biotechnology 4 : 1001-1004 (1986) and Klein et al., Nature 327 : 70-73 (1987). In each case, the transformed cells are regenerated into whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for the transformation of dicotyledonous plants because of its high efficiency transformation and widespread utility for many different species. Many crop species commonly transformed by Agrobacterium include tobacco, tomatoes, sunflowers, cotton, rapeseed, potatoes, soybeans, alfalfa and poplars. EP 0 317 511 (cotton), EP 0 249 432 (tomato), WO 87/07299 (Brassica) or US Pat. No. 4,795,855 (Poplar). Agrobacterium transformation typically involves a host vector that can vary depending on the complement of the vir gene carried on a co-stable Ti plasmid or a binary vector carrying the desired foreign DNA (eg pCIB200 or pCIB2001). To strains (e.g., strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5 : 159-169 (1993))). The delivery of the recombinant binary vector to Agrobacterium is carried out with E. coli carrying the recombinant binary vector. E. coli, a helper E. carrying a plasmid such as pRK2013 and capable of transferring a recombinant binary vector to a target Agrobacterium strain. Achievement is achieved by trimeric crossing process using E. coli strains. Alternatively, the recombinant binary vector can be delivered to Agrobacterium by DNA transformation. Hofgen & Willmitzer, Nucl. Acids Res. 16 : 9877 (1988).

Transformation with recombinant Agrobacterium of the target plant species typically involves co-culture of Agrobacterium with explants from the plant and is in accordance with protocols well known in the art. Transformed tissue is regenerated on a selectable medium bearing the antibody or herbicide marker resistance marker present between the binary plasmid T-DNA boundaries.

Transformation of dicotyledonous plants can also be carried out using bioistic (bioistic). Particularly preferred for soybean transformation is the method described in US Pat. No. 5,024,944.

Transformation of monocotyledonous plants

Transformation of most monocotyledonous plant species is currently a common means. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques and particle bombardment into callus tissue. Such transformations can be performed using single DNA species or multiple DNA species (ie, simultaneous transformation), both of which are suitable for use with the present invention. Simultaneous transfection has the advantage of being able to generate transgenic plants with locus unlinked to the gene of interest, which avoids complex vector construction and allows the removal of selectable markers in subsequent generations that are considered desirable. Can have However, a disadvantage of using co-transfection is that the frequency of integration into separate DNA species genomes is less than 100%. Schocher et al. Biotechnology 4 : 1093-1096 (1986)].

Patent applications EP 0 292 435, EP 0 392 225 and WO 93/07278 describe the preparation of callus and protoplasts from selected suburban corn, transformation and transformation of protoplasts using PCG or electroporation. Techniques for the regeneration of corn plants from isolated protoplasts are described. See Gordon-Kamm et al. Plant Cell 2 : 603-618 (1990) and Fromm et al. Biotechnology 8 : 833-839 (1990) discloses a technique for the transformation of A188-derived maize strains using particle bombardment. See also WO 93/07278 and Koziel et al. Biotechnology 11 : 194-200 (1993) describes a technique for the transformation by particle bombardment of selected suburban corn. This technique uses 1.5-2.5 mm long immature corn embryos excised from corn ears 14 to 15 days after pollination and the PDS-1000He Biolistics device for impact. Corn is also described, for example, in Ishida et al., 1996; High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens, Nature Biotechnology 14, 745-750 can be transformed by Agrobacterium.

In addition, transformation of rice may be performed by direct gene transfer techniques using protoplasts or particle bombardment. Protoplast-mediated transformation methods are described for the saponica and indica types in Zhang et al., Plant Cell Rep 7 : 379-384 (1989); Shimamoto et al. Nature 338 : 274-277 (1989); Datta et al. Biotechnology 8 : 736-740 (1990). Both types are also typically transformable using particle bombardment. See Christou et al. Biotechnology 9 : 957-962 (1991).

Patent application EP 0 332 581 describes a technique for the generation, transformation and regeneration of Poideae protoplasts. These techniques enable the transformation of Dactylis and wheat. In addition, wheat transformation into type C long term regenerable callus cells using particle bombardment is described by Vasil et al. Biotechnology 10 : 667-674 (1992), and wheat transformation methods of immature embryos and immature embryo-derived callus using particle bombardment are described in Weeks et al. Plant Physiol. 102 : 1077-1084 (1993). However, preferred techniques for wheat transformation include transforming wheat by particle bombardment of immature embryos, and include a high sucrose or high maltose step prior to gene transfer. Prior to impact, any number of embryos (0.75-1 mm long) were subjected to 3% sucrose (Murashige & Skoog, Physiology Planetarium 15 : 473-497 (1962)) and 2,4 for the induction of somatic embryos that should proceed under cancer conditions. Plate on MS medium containing -D 3 mg / L. On selected days during the impact, embryos are removed from the induction medium and placed on osmoticum (ie, induction medium containing sucrose or maltose at the desired concentration, typically 15%). Embryos are protoplasted for 2-3 hours and then shocked. Twenty embryos per target plate are typical, but not critical. Using standard methods, suitable gene-bearing plasmids (eg pCIB3064 or pSG35) are precipitated onto micrometer sized gold particles. Embryos of each plate are launched with a DuPont Biolistics ^R helium device using ~ 1000psi burst pressure using a standard 80 mesh screen. After the impact, the embryos are placed back under dark conditions and recovered for about 24 hours (still on osmoticum). After 24 hours, embryos are removed from Osmotichum and placed on induction medium to be left for about 1 month before regeneration. After about one month, embryo explants containing embryogenic callus were regenerated medium (MS + 1 mg / ml) further containing suitable selection agents (10 mg / l Basta for pCIB3064 and 2 mg / l methotrexate for pSOG35). 1 NAA, 5 mg / L GA). After about one month, the developed projectiles are transferred to a larger sterile vessel known as "GA7s" containing a selection agent at a concentration equal to 1/2 concentration MS, 2% sucrose. Patent application WO 94/13822, which is incorporated herein by reference, describes a method for wheat transformation.

Chromosome Transformation

Chromosome transformation techniques are described in US Pat. Nos. 5,451,513, 5,545,817 and 5,545,818; PCT Patent Applications WO 95/16783 and WO 98/11235; And McBride et al. (1984) Proc. Natl. Acad. Sci. USA 91, 7301-7305, the entire contents of which are expressly incorporated herein by reference. Basic techniques for chloroplast transformation include biomarkers or protoplast transformation (e.g., calcium chloride or PCG mediated transformations), clonal chromatin DNA flanking using a target gene suitable for the region of interest with the desired gene. It includes introducing into. 1 to 1.5 kb flanking regions, called targeting sequences, facilitate homologous recombination with the chromatin genome, allowing substitution or modification of specific regions of the plasma. Initially, point mutations in the chloroplast 16S rRNA and rps12 gene that provide resistance to spectinomycin and / or streptomycin are used as selectable markers for transformation. See Svab, which is incorporated herein by reference. Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530 and Staub, J.M., and Maliga, P. (1992) Plant Cell 4, 39-45. This results in about one stable homologous plasma transformant per 100 impacts of the target leaf. The presence of cloning sites between these markers allows the generation of chromatin targeting vectors for introduction of foreign genes. See, eg, Staub, JM, and Maliga, P. (1993) EMBO J. 12. , 601-606. Substantial increase in transformation frequency is achieved by substitution of a recessive rRNA or r-protein antibody resistance gene with a dominant selectable marker, a bacterial aadA gene encoding spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase. Obtained by Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917. Previously, such markers have been used successfully for high frequency transformation of the chromatin genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991), incorporated herein by reference). Nucl. Acids Res. 19, 4083-4089. Other selectable markers useful for chromatin transformation are known in the art and are included within the scope of the present invention. Typically, about 15 to 20 cell division cycles following transformation are necessary to reach homologous state.

Chromosome expression, in which genes are introduced throughout thousands of copies of the circulating chromatin genome present in each plant cell by homologous recombination, yields a huge copy number advantage over nuclear-expressed genes, resulting in 10% of the total soluble protein. Allow expression levels that can easily be exceeded. However, these high expression levels can cause potential growth problems, especially during early plant growth. Similar problems are caused by the expression of bioactive enzymes or proteins that can be very detrimental to the survival of the transgenic plant and, if constitutively expressed, may not be successfully introduced into the plant genome. Thus, in certain aspects, the present invention is directed to the expression of tobacco chemically inducible PR-1a promoters of tobacco in the nuclear genome of chloroplast-targeted phage T7 RNA polymerase (see US Pat. No. 5,614,395, incorporated herein by reference). Under control it is coupled to the chloroplast reporter transformed gene regulated by the T7 gene 10 promoter / terminator sequence. For example, when a pigment transformant homologous to the uidA gene inherited from a parent encoding a β-glucuronidase (GUS) reporter is pollinated by a lineage expressing T7 polymerase in the nucleus F1 plants are obtained that retain both transgenic constructs that do not express the GUS protein. Synthesis of large amounts of enzymatically active GUS is initiated in the plastids of the plant only after leaf application of PR-1a inducer compound benzo (1,2,3) thiadiazole-7-carbotinic acid S-methyl ester (BTH) do.

Brief description of the sequences in the sequence listing

SEQ ID NO: 1 protein sequence of thioredoxin from Metanococcus jannaschii

SEQ NO: 2: protein sequence of thioredoxin from Arcaeoglobus pulgidus (trx-1)

SEQ ID NO: 3 protein sequence of thioredoxin from Arcaeoglobus pergidus (trx-2)

SEQ NO: 4: protein sequence of thioredoxin from Arcaeoglobus pulgidus (trx-3)

SEQ ID NO: 5: protein sequence of thioredoxin from Arcaeoglobus pulgidus (trx-4)

SEQ ID NO: 6 protein sequence (trxB) of thioredoxin reductase from Metanococcus jannaschii

SEQ ID NO: 7 protein sequence of thioredoxin reductase from Arcaeoglobus pulgidus (trxB)

SEQ ID NO: 8: Clontech Sequence

SEQ ID NO: 9: Josh Sequence

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

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and described in Sambrook et al. (1989) Molecular Cloning and Ausubel et al. (1994) Current Protocols in Molecular Biology.

Example 1 Transformation of Corn into Thermostable Thioredoxin

Genes expressing heat stable thioredoxin derived from Metanococcus zanarisky with the sequence of MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPQKAMEYGIMAVPTIVINGDVEFIGAPTKEALVEAIKKRL (SEQ ID NO: 1) were used to express a gamma-codon-specific species using a gene as described in US Pat. No. 5,625,136. Prepared under the control of an expression cassette introduced between the promoter and the T-DNA border of the pGIGUP plasmid. The T-DNA of this plasmid is a bar gene expressible in plants driven by the ubiquitin promoter that provides resistance to phosphinothricin (Christensen et al., Plant Mol. Biol. 18: 875-689, 1992]. The plasmid also contains an octopin synthase that activates the sequence having the region of the N-terminal codon (mannopine synthase gene) of a coding gene (SMAS) driven by a chimeric promoter derived from the octopin and mannino synthase genes. Trimer upstream of the promoter; see Ni et al., Plnt J. 7: 661-676, 1995) and contain the GUS gene (beta-glucuronidase). This intron-GUS gene expresses GUS activity in plant cells but not in Agrobacterium. Alternatively, thermally stable thioredoxin derived from Metanococcus xannaskii is cloned into plasmid pNOV117 containing pmi genes expressible in plants driven by the selection corn ubiquitin promoter on mannose. Christensen et al. 1992, Joersbo et al., 1998].

Strain A. Tumefaciens LBA4404 (pAL4404, pSB1) is used in this experiment. pAL4404 is a released helper plasmid. pSB1 is a broad host plasmid containing 15.2 kb KpnI fragments from the homology region for pGIGUP and pNOV117 and the virulence region of pTiBo542 (Ishida et al., 1996; High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens, Nature Biotechnology 14, 745-750]. Introduction of the plasmid pGIGUP or pNOV117 into LBA4404 (pAL4404, pSB1) by electroporation results in the mutual integration of pGIGUP or pNOV117 with pSB1. The T-DNA of pNOV117 contains the mannose-6-phosphate isomerase gene driven by the ubiquitin promoter that provides the ability to metabolize mannose and the thioredoxin gene described above.

Agrobacterium for 3 days on YP medium (5 mg / l yeast extract, 10 g / l peptone, 5 g / l NaCl, 15 g / l agar, pH 6.8) supplemented with 50 mg / l spectinomycin and 10 mg / l tetracycline To grow. To collect the bacteria as a loop, 10 ⁹ to 5x10 ⁹ then suspended in N6 liquid medium at a density of cells / ml. Agrobacterium cells are also collected from overnight cultures in YP medium and resuspended in N6 liquid medium. For 1 L of medium, 4 g of powdered N6 salt (Sigma, St. Louis, MO), sucrose 30 g, myo-inositol 100 mg, glycine 2 mg, thiamine 1 mg, pyrotoxin HCL 0.5 mg, nicotinic acid 0.5 mg, 2,4-D 2 mg [from a stock solution (1 mg / mL) prepared by dissolving 2,4-D in dilute KOH) is added. Adjust to pH 6.0 with 1M KOH, add 3 g of Gelrite and autoclave.

Corn immature embryos are obtained about 10 to 14 days after self pollination. About 25 immature conjugated embryos are distributed per plate to each other plate containing medium capable of inducing and supporting embryogenic callus formation.

Immature embryos are seeded on a plate containing Agrobacterium with a Ti plasmid containing a selectable marker gene or in a liquid. Immature embryos are plated on callus initiation medium containing silver nitrate (10 mg / L) before or immediately after inoculation with Agrobacterium. About 25 immature embryos are placed on each plate. 16 to 72 hours after inoculation, immature embryos are transferred to callus initiation medium containing silver nitrate and cytotaxy. Selection of transformed cells is performed as follows:

Mannose is used to select transformed cells in vitro. These choices can be applied as little as 1 g / L on days 2-20 after inoculation, and maintained for a total of 2-12 weeks. Embryonic callus thus obtained is regenerated on the standard regeneration medium in the presence or absence of mannose. Whole plants are tested by the chlorophenol red (CR) test for resistance to mannose. This assay uses a pH sensitive indicator dye to indicate whether cells have grown in the presence of mannose. Growing cells produce a pH change in the medium, changing the color of the indicator chlorophenol red (CR) from red to yellow. Plants that exhibit resistance to mannose are readily identified by this test. Plants with a positive response by the CR test are analyzed by PCR for the presence of the mannose gene. Analyze by Southern blot using plants with positive response to PCR test.

Regenerated plants are analyzed for expression of thioredoxin. The plant is developmentally normal. Corn grains from progeny plants derived from the highest expression events are analyzed in a small scale wet grinding process and starch extractability is measured in comparison to corn of the same genotype without the thioredoxin transgene. Corn expressing the thioredoxin gene shows substantially greater starch availability in the wet milling process than untransformed corn of the homologous gene.

Example 2: Transformation of Corn with Heat Stability Thioredoxin and Thioredoxin Reductase

Using the procedure described in Example 1, corn was described above. Cotransforms with genes for both thioredoxin and thioredoxin reductase from Jannasquiy. Both genes are placed under the control of a seed specific gamma agent promoter. The two genes are linked and placed between the left and right boundaries of the pGIGUP or pNOV117 plasmids, enhancing the likelihood that the two genes can be inserted into the plant's chromosome as a single insert.

Regenerated plants are analyzed for expression of thioredoxin and thioredoxin reductase. The plant is developmentally normal. Corn grains from progeny plants derived from the highest expression events were analyzed in a small scale wet grinding process and starch extractability was measured in comparison to corn of the same genotype without the thioredoxin / thioredoxin reductase transgene. do. Maize expressing thioredoxin and thioredoxin reductase genes exhibit substantially greater starch availability in wet milling processes than untransformed maize of homogeneous genes.

Claims (13)

Soaking the grains at elevated temperature in the presence of supplemental thioredoxin and / or thioredoxin reductase, and A method of enhancing starch and protein separation efficiency in a grain grinding method comprising the step of separating the starch and protein components of the grain. The method of claim 1, wherein the grain comprises grain from a transgenic plant wherein the transgene expresses thioredoxin and / or thioredoxin reductase. The method of claim 2 wherein the plant is selected from maize (Zea mays) and soybeans. A plant comprising a heterologous DNA sequence encoding thioredoxin and / or thioredoxin reductase, which is stably integrated in nuclear or chromatin DNA. The plant of claim 4 wherein the thioredoxin and / or thioredoxin reductase is thermally stable. The plant of claim 5, wherein the 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: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. The plant according to any one of claims 4 to 6, which is selected from corn and soybeans. An expression cassette expressible in a plant comprising a coding region for thioredoxin and / or thioredoxin reductase operably linked to a promoter and terminator sequence acting in the plant. The expression cassette of claim 8, wherein the thioredoxin and / or thioredoxin reductase is heat stable. The plant-expressable expression of claim 9, wherein the 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: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. cassette. A method for producing a grain comprising high levels of thioredoxin and / or thioredoxin reductase, comprising transforming a plant with an expression cassette according to any one of claims 8 to 10. A first plant comprising a heterologous expression cassette comprising a promoter that is regulated and regulated by a transcriptional activator operably linked to a DNA sequence encoding thioredoxin and / or thioredoxin reductase is regulated with the transcriptional activator. Pollination with a pollen from a second plant comprising a heterologous expression cassette comprising a promoter operably linked to a DNA sequence encoding a transcriptional activator capable of regulating a promoter; and A method for producing a grain comprising high levels of thioredoxin and / or thioredoxin reductase, comprising recovering the grain from the pollinated plant as in the above step. Use of a plant or plant material according to any one of claims 4 to 7 as an animal feed.