UA75562C2 - Methods and compositions for modification of the level of secondary metabolic compounds in plants - Google Patents

Abstract

The present invention provides a method of making a genetically transformed plant which has an altered content of at least one product of a secondary metabolic pathway. The method consists of introducing into a plant cell capable of being transformed and regenerated to a whole plant a DNA expression cassette. The expression cassette includes DNA sequences required for transformation and selection in plant cells. It also includes a DNA sequence that, under the control of a promoter active in plant cells, encodes a protein capable of modifying the utilization of a substrate in the secondary metabolic pathway. The substrate is not a primary metabolite of the group selected from glucose, amino acids, common fatty acids and nucleotides. A plant or plant tissues including seeds can then be recovered having an altered content of at least one product of the secondary metabolic pathway. The invention also provides for feed products derived from the plants and seeds obtained according to the method.

Description

Description of the invention

This application is a continuation-in-part of US Application Serial No. 09/012,453 and claims priority from the earlier application 60/072,156.

The present invention proposes methods and compositions for changing the level of compounds formed as a result of the processes of secondary metabolic pathways in plants. The invention also provides plant cells with an altered level of secondary metabolites and plant seeds with an altered level of secondary metabolites. In one embodiment, the level of non-nutritive secondary 70 metabolic substances is changed in plants, plant cells and plant seeds according to the invention. In another embodiment, the level of products of secondary metabolic pathways, such as phenylpropanoids and sugar alcohols, is altered in plants, plant cells and plant seeds according to the invention. The invention also provides genetic constructs and vectors used to modify the level of secondary metabolites in plant cells and seeds. The invention also relates to flour from modified grain, animal feed containing flour from modified grain, and in particular flour from grain in which the level of secondary 12 metabolites is reduced or changed.

Plants produce a large number of different compounds through secondary metabolism. As a result of secondary metabolic pathways, which are not considered essential for plant metabolism, unique biochemical substances are often formed, and some of them are considered non-nutritive or even toxic. Secondary metabolic pathways and compounds resulting from these pathways are often species or genus specific.

Therefore, the processing of secondary metabolic pathways can lead to the emergence of new compositions of biochemical substances or plant tissues with a changed level of secondary metabolites. In particular, manipulation of secondary metabolic pathways to alter the levels of secondary metabolic compounds that are non-nutritive or toxic in nature may lead to unique applications in the food and feed industry.

Processing of secondary metabolism should preferably be carried out without changing the biochemical processes that are considered vital for the growth and viability of the plant cell. A set of biochemical processes and compounds, (which are important for the growth and viability of plants, which participate in them, are considered the processes of the main metabolism and their products. The main metabolism is generally considered as such, which includes those biochemical processes that lead to the formation basic sugars (for example, glucose), amino acids, conventional fatty acids, nucleotides and their polymers (polysaccharides such as starch, proteins, lipids, ribonucleic acid and deoxyribonucleic acid, etc.) Zesopdagu "Fa

Meiaboizt ip Siyigea Riapi SeiYiv, Mem Rpuyuiodivi, 134:553-569, 1996).

Thus, it is customary to distinguish primary metabolism as a process of metabolism that is important for the viability and growth of all plant cells, and secondary metabolism as biochemical processes that are not vitally necessary for all plant cells. For example, secondary metabolic pathways determine such characteristics of a plant as color, taste, morphology, etc. Also, as a result of the processes of secondary metabolic pathways, various compounds are formed that are perceived by insects or participate in the reaction to pathogens. Some of these compounds may be beneficial to some plant species in the wild, but in the cultivated state these compounds may degrade the quality of the cultivated product or limit the use of the crop in certain applications. "

Some secondary metabolites are unique compounds that have evolved in species as a result of 50 specialized biochemical processes. Secondary metabolism is not characterized by an excess of biochemical mechanisms, which is typical for primary metabolism, therefore, as a rule, products of secondary metabolism are not

"Iz" are formed in the plant in many ways. Secondary metabolites are typically more specific to individual plants than the common biochemicals involved in major pathways.

Numerous attempts to modify the processes of basic metabolism have led to the appearance of plant cells with altered levels of starch or oil (lipids). However, a gross effect on the basic metabolism can lead to this. harmful consequences. For example, one can change the composition of lipids, but removing lipids would be a negative 4! consequence for cell viability. Treatment of basal metabolism is not always a successful goal because a large number of biochemical mechanisms can overcome any treatment attempts. Thus, the processes of primary and secondary metabolism in plants are often difficult to modify in a way that would be fully predictable and obtain 20 applicable and noticeable results under cultivation conditions.

In some cases, the underlying metabolism has been successfully modified to produce a new phenotype in which a change in composition rather than a reduction or elimination of a single substance has been achieved. Typically, such manipulations were carried out by ectopic expression of a plant gene, for example, by overexpressing a gene in some tissues or in a constitutive form more often than in a regulated form, or by inhibiting the activity of a certain gene using antisense RNA, ribosomal enzymes, or cosuppression. However, it was very difficult to predict a

GF) rhyogenic consequences of such manipulations.

Plant enzyme expression can be modified at many levels. These include control at the level of gene expression, translation, protein processing, and allosteric control of protein function. Thus, ectopic expression of a plant gene involved in basic metabolism may not overcome complex 60 biochemical control of basic metabolism. In addition, the problem of a large number of major metabolic pathways is also difficult to overcome, since major metabolic processes are important for plant growth and viability. Thus, attempts to alter the underlying metabolism often fail to produce the desired phenotype. Moreover, tests of such modified plants in the field or in extreme environmental conditions often reveal that the intended effect is not observed or the plant's viability is compromised. Thus, modification of core metabolism requires careful consideration of the core metabolic process or judicious implementation of a step within the pathway to achieve the desired phenotype.

Manipulation of the processes of secondary metabolic pathways has been hampered by insufficient understanding of their biochemistry, insufficient information about the genes involved in secondary metabolic pathways, and the complex interaction between biochemical processes in general.

However, methods of altering secondary metabolism may provide useful means to create new phenotypes, including phenotypes with altered amounts of secondary metabolism compounds, such as those considered non-nutritive in nature. Therefore, the secondary metabolic pathways of substances are an important task for 7/0 genetic modification of plants.

Attempts were made to transfer the path of betaine biosynthesis to plants that are incapable of synthesizing betaine, an osmoprotectant. (INoitvigot, K.O. sei aiYi., Rgodisiop oi (She EzsPpegispia soii Beiaipe-aidepude depudgodepaze? Ap eplute gedytsiged yog zupipeviz oi oztorgoiesiapi diusipe Breiaipe, ip (gapzdepis riapiv, Te

Riapi docsgpa!i, (1994) 6(5):749-58| describes the use of the gene encoding betaine-aldehyde dehydrogenase for the 7/5 synthesis of the osmoprotectant glycine betaine.

The work |OSegmepi arzigashi AM 96-512578, Touoia didozpa KK, October 15, 1996) describes the use of the choline dehydrogenase gene and the betaine-aldehyde dehydrogenase gene for the synthesis of the betaine osmoprotectant.

Two biochemical pathways in plants, considered as pathways of secondary metabolism, have been the subject of research aimed at changing the level of the end product. The methods used to manipulate these pathways did not lead to the desired result. For example, the phenylpropanoid pathway is involved in the formation of lignin and can be considered a secondary metabolic pathway. Lignin biosynthesis is part of the general pathway of phenylpropanoid biosynthesis, in which at least three main phenolic precursors are formed: coumaric, ferulic, and sinapic acids, the products of which are polymerized into lignin and other phenolic compounds (see Fig. 2). high school

In attempts to alter the secondary phenylprvlanoid metabolic pathway, the genes for many enzymes involved in the formation of lignin monomers are now being considered as targets for reducing lignin levels through the use of antisense sequences or cosuppression (eg, US Pat. No. 5,451,514, US Pat.

Mo5633439, MO 93/05160, MUO 94/08036). These target genes include those encoding cinnamyl alcohol dehydrogenase, caffeic acid O-methyl-transferase, and phenylalanine ammonium lyase. These methods are aimed at reducing the level of lignin, as it is believed that this will have an overall beneficial effect on plant processing or assimilation. ise)

However, reducing the level of lignin by using an antisense sequence or cosuppression of one of the genes of the phenylpropanoid pathway can have a large number of undesirable consequences. These include: increased susceptibility to diseases, changed growth rates or a decrease in the physical strength of plant fibers and, as a result, a decrease in agronomic efficiency. It was shown that the inhibition of the enzyme phenylalanine ammoniolyase leads to the emergence of numerous harmful phenotypes (EiKipa ei. aiI., Abpogtai Riapi

Oemeyuortepi apa ОЮomp-Kedshayop oi RVPepuIrgorapoiiiy Viozupiyeviv ip Tgtapzdepis Torasso Sopiaipipd a

Neyegoisdoiz RNepuiaiapipe Attopia Iuaze Sepe, Rgos. May Asai. si. OBA 87:9057-9061, 1990). The enzyme phenylalanine ammoniolyase acts on the main metabolite phenylalanine, which is an amino acid. The results of these experiments show that altering secondary metabolism by modifying one of the main metabolites, which is involved in a separate secondary metabolic pathway, can create unwanted and harmful phenotypes.

Therefore, the choice of biochemical step or steps within the secondary metabolic pathway is crucial for; creating plants that are phenotypically normal but exhibit reduced levels of a specific secondary metabolite.

In addition, the use of antisense RNA or cosuppression may not result in a decrease in the level or -I specificity of a particular secondary metabolite that is economically acceptable. In addition, inhibition of genes encoding key enzymes can affect the expression of related genes. Thus, little progress has been made in reducing the levels of phenolic compounds considered non-nutritive or in reducing c-lignin by using antisense RNA or cosuppression without harmful side effects.

A second example of attempts to change a metabolic pathway that have failed is the modification of biosynthesis

Me, glucosinolate in rapeseed. Attempts to alter glucosinolate levels in rapeseed meal have been reported

With the way of processing -- the glucosinolate pathway. A method of changing the biosynthesis of glucosinolate was proposed, which consisted in creating a new pathway competitive with sulfur, which is used in the formation of glucosinolates, or in reducing the level of tryptophan, which is used in the formation of glucosinolates by converting tryptophan into tryptamine. ", Ru

Yogapit, Spamade| 5 YuOe Iisa, ribiizpei ip: Sepegis epdipeegipd oi riapi zesopdagu teiyaroijist 1994, Riepyt

F) Ryviizpipd Sogrogayop; Meme ZhMogk; OA. However, this method was not successful in reducing glucosinolate levels in canola flour. It turned out that this method cannot reduce the level of non-food glucosinolates in rapeseed flour. Because glucosinolates are formed in the leaves of plants and then transferred to the seeds. Therefore, this method was based on the assumption that a simple change in the availability of one of the main metabolites (sulfur, amino acid tryptophan) involved in the formation of glucosinolates will reduce the formation of glucosinolates. However, the main glucosinolates in the seed are aliphatic glucosinolates that do not use the amino acid tryptophan to create side chains.

Moreover, the results of these experiments (for example, (Spamade | ei. aiYi., Rgos. Mai. Asai. Zsi. O5A, 65 91:2166-2170, 19941) showed that transgenic plants, which contain an enzyme capable of changing the amount of the basic amino acid tryptophan, do not have reduced amounts of glucosinolates in seeds; the level of aliphatic glucosinolate in seeds is equal to or even greater than in non-transgenic plants. Thus, the total formation of glucosinolates in seeds was not reduced, even when the level of the minor component (indole glucosinolates) was reduced. Genetic studies show that plants with low glucosinolate levels

Can be obtained by ordinary dilution. But there are a large number of loci that control the low level of glucosinolates in cruciferous plants. There are numerous biochemical transformations that occur in the biosynthesis of glucosinolates, and these transformations, like all or most processes of glucosinolate synthesis, must be targeted in a way to reduce total glucosinolate levels. Thus, when developing a general method of changing the formation of glucosinolates in cruciferous plants, it is necessary to take into account various enzymes and 7/o substrates involved in the biosynthesis of glucosinolates.

However, it turns out that the enzymes used to produce this modification also affect the main metabolites (the amino acid tryptophan and the mineral sulfur) and therefore any significant change in the level of these compounds in the plant cell can cause harmful effects. Thus, the proposed method cannot specifically affect secondary metabolism. Indeed, a change in tryptophan levels can lead to many harmful effects. Thus, changing the level of the main metabolite does not have the desired effect of reducing glucosinolates in rapeseed meal, which emphasizes the complexity of changing the main metabolism.

In MO 97 23599 A Meiyipoa tog Kedshaiyop oi Riapi Sotroziyop, (E.I. OyRopi apa Rigatse Kezeagsp Roshpaayop,

Since July, 1997| describes the use of ferulate-5-hydroxylase (FG) obtained from a cruciferous plant to regulate the level of lignin in plant cells. The FG enzyme described in UMO 97 23599 is an enzyme generally considered to be part of the phenylpropanoid pathway in plant cells, such as the cruciferous plant from which it was derived. The FG enzyme described in UUO 97 23599 does not show activity in the transformed cell, but shows activity in the cell from which it was obtained.

Thus, the general method of changing secondary metabolism will be useful for changing the biochemical composition of plant tissues. It may include, for example, reducing the level of non-nutritive compounds, changing the profile of secondary metabolic substances, obtaining plant tissue with altered processing, changing the level of compounds that are used in industry or in pharmaceuticals, creating plants with a changed taste, structure i) or appearance, creating plants with altered secondary metabolites that attract insects associated with disease susceptibility or other biological processes affected by secondary metabolites, or plants with positively altered growth characteristics due to altered secondary metabolite levels. The present invention proposes a method of directed influence on the formation of secondary metabolites. The method includes changing the availability of the substrate, which is specific to the secondary metabolic pathway and important for the formation of end products of secondary metabolism, in particular, compounds participating in 1--5 biochemical stages of the formation of the end product. Directing the impact on substrates at stages close to the formation of the final product allows you to avoid problems associated with changes in the level of metabolites, which are also involved in the main pathways, since it occurs after the substrate enters the main metabolic pathway. Thus, this method offers new means of specifically directing the effect of reducing or changing the level of secondary metabolites by identifying precursors involved in the secondary metabolic pathway, which are not substrates of the main metabolic pathways.

The method can also include changing the availability of the substrate in a tissue-specific way so that only certain tissues, for example, seed tissues, will be changed. в с Thus, this method offers new means of specifically directing the impact on reduction

Or a change in the level of secondary metabolites due to the detection of precursors participating in the secondary metabolic pathway, which are not compounds of the main metabolism.

In the first embodiment, this invention offers a method of creating a genetically transformed plant, consisting of: -And A) introducing an expression cassette into a cell that can be transformed and regenerated into a whole plant

DNA containing, in addition to the DNA sequence required for transformation and selection in plant cells, also 1 DNA sequence, which, under the control of a promoter active in plant cells, encodes a protein capable of changing the use of a substrate in a secondary metabolic pathway, provided that the substrate is not is a major metabolite such as glucose, amino acids, conventional fatty acids, and nucleotides;

Me, B) growing a plant with an altered level of at least one of the products of the secondary metabolic pathway.

Ge In another embodiment, this invention provides a method of creating genetically transformed seeds, which consists of growing the plant obtained in steps A and B of the method described above under the conditions of seed formation.

Recombinant DNA is a DNA sequence integrated into the chromosome and genome of a fertile plant in such a way that it is inherited by subsequent generations.

F) In another embodiment, the present invention provides vectors for plant transformation, plants and seeds transformed according to the method described above, and food products containing seeds or meal obtained from seeds. in Brief description of drawings:

Fig. 1: Schematic representation of the general scheme of the method of changing any secondary metabolic pathway.

Fig2: Schematic representation of general phenylpropanoid metabolism and sinapin formation in crucifers.

Fig. 3: Beginning and development of sinapin synthesis in germinating seeds. Analysis of seeds of Vgazvzis vapors variety 65 UUeviag by thin-layer chromatography.

Fig. 4: Quantitative analysis of the accumulation of sinapin in germinating seeds by the NO PO method.

Fig. 5: Determination of the ability of germinating seeds to synthesize sinapin by injecting radioactive choline through the stem of excised pods and incorporating the label into sinapin from the 7th to the 43rd day after pollination.

Fig. 6: Determination of the ability of germinating seeds to synthesize sinapin by absorption of a solution containing radioactive choline by the isolated seed and inclusion of a label in sinapin from the 43rd to the 64th day after pollination.

Fig. 7: Accumulation of newly synthesized sinapin in the cotyledon, embryo axes and seed coat of germinating seeds as a fraction of the total amount of labeled sinapin in the seed.

Fig. 8: The level of sinapin in the cotyledon and axils of the germinating seed embryo per unit mass of the tissue sample.

Fig. 9: Determination of the level of sinapin in the cotyledon and the axis of the embryo of a germinating seed per seed, 7/0 ie per axis or a pair of cotyledons.

Fig. 10A and 10B: Nucleotide sequence of the open reading frame of choline oxidase (EO). IU Mo3).

Fig. 11: Predicted sequence of amino acids of the open reading frame of choline oxidase (EO). IU Mod).

Fig. 12: Diagram of the vector pH 731, containing the XO gene under the control of a tissue-selective promoter, for plant transformation.

Fig. 13: Reduction of the level of sinapin in the seeds of Vgazvzis zr. due to the expression of the XO gene.

Fig. 14: Diagram of the vector pH5 981, containing the BADH gene under the control of a tissue-selective promoter, for plant transformation.

Fig. 15: Reduction of the level of sinapin in the seeds of Vgazvis zr. due to the expression of the XO and BADH genes.

Fig. 16: Change in the level of phenolic compounds in the seeds of Vgazzis zr. due to the expression of the XO and BADH genes.

Fig. 17: Nucleotide sequence of synthetic Fedulic acid decarboxylase gene of B. rhytii, optimized for expression in plant cells (ZEO). 1I0.Mo1).

Fig. 18: Estimated sequence of amino acids of the protein encoded by the synthetic open reading frame of ferulic acid decarboxylase B. ritiiv5 (ZEO. IU. Mo2).

Fig. 19: Restriction map of the vector containing the Fedulic acid decarboxylase gene under the control of constitutive promoter 355 for plant transformation.

Fig. 20: Restriction map of the vector containing the Fedulic acid decarboxylase gene under the control of i) seed-selective napin promoter for plant transformation.

Fig. 21: Accumulation of phytic acid during seed germination.

Fig. 22: Features of the accumulation of phytic acid in various tissues of germinating seeds. Fig. 23: Level of assimilated labeled myoinositol in phytic acid, lipids, TPA-soluble fraction of cell wall and cell debris. X,

Fig. 24: Sequence of the amplified DNA fragment of the myoinositol O-methyltransferase (ZEO) gene. IU Mob). yu

Fig. 25: Restriction map of the pZIMT vector containing the cassette: promoter 355 - IMT-5Y5-Moz terminator, in pEO400. at

Fig26: Restriction map of pMIMT vector containing the seed-selective promoter 355 -IMT-Moz i- terminator, in pKO400.

Fig. 27: RSC analysis of transgenic plants containing the BMI gene.

Fig. 28: Northern blot analysis of plants containing the BMI gene.

Fig. 29: Diagram of the observed decrease in the level of phytic acid in transgenic plants. "

Fig. 30: Reduction of the level of phytic acid in HE1, E2 and EZ plants containing the pZIMT vector. from c Fig. 31: Reduction of the level of phytic acid in E1 and E2 plants containing the RMIMT vector.

This invention relates to a specific reduction or change in the ratio of the level of precursors and products;" secondary metabolism, different from the main metabolites. In this way, the potential harmful effects of changing the main metabolic pathways to achieve a similar result can be avoided. Thus, this one

The invention does not relate to compounds that could be considered major metabolites. -I In the best embodiment, the availability of the substrate is changed due to the use of an enzyme, for example, foreign to this plant cell, suitable for acting on this substrate and such that removes the said substrate or changes its availability in the available set of substrates. A general overview of the method and interaction between primary and secondary metabolism is shown in Fig.1. The use of a foreign enzyme to change the 5p flow of products of the secondary metabolic pathway leads to modification of the desired final secondary

Me, a metabolite. This alters the flow of products in the secondary metabolic pathway, leading to a modification of the desired

From the final secondary metabolite. Thus, the level of the final product at certain enzymatic stages of the secondary metabolic pathway decreases, while at other stages, on the contrary, the product accumulates, inhibiting the enzymes responsible for the formation of this product. This feedback inhibition, in turn, affects the formation of the final product of the entire secondary metabolic pathway. Therefore, a change in the level of products of a certain secondary metabolic pathway and related processes is achieved

F) due to the use of new enzymes that change the flow of biochemical substances (ie, substrates and their ka products) through this pathway. Foreign enzymes have been expressed in plant tissues, causing metabolic redistribution of precursors within these pathways to substances that accumulate without harmful effects in the plant cell.

In some embodiments, as a result of using this method, useful products or products that have a beneficial effect on the plant cell are formed.

In one embodiment, the target precursor is altered by the addition of a new enzyme. In one preferred aspect of the invention, an enzyme is selected that has an activity foreign to the plant cell in which it is expressed. For example, such an enzyme in its normal state is not associated with the secondary metabolic pathway considered in a plant cell. The enzyme used can be of plant, animal or microbiological origin and can be modified for proper expression in plant cells.

The selected foreign enzyme is able to modify the precursor in such a way as to change its availability in the formation of the secondary metabolite. Using an enzyme foreign to the cell in which it is expressed,

It is possible to bypass the usual biochemical control mechanisms and change the secondary metabolism in a predictable direction. The modification of the products of the secondary metabolic pathway related to the secondary metabolic pathway of sugar alcohols and phenylpropanoids is of particular interest within the scope of this invention.

Additional examples of the use of this method are changes in the level of modified sugar compounds such as galactose and non-food sugar glycosides such as stachyose and raffinose. Galactose is converted to /o hapactinol, which is one of the precursors of the formation of these non-food sugar glycosides. The precursor of galactose is the conjugated form -- UDP-galactose. As a method, within the scope of the present invention, it is possible to consider preventing the formation and accumulation of UDF-galactose (and its subsequent transformation into galactinol) due to the use of the activity of an enzyme foreign to plant cells, and this enzyme is capable of changing the level of UDF-galactose. UDP-galactose 4-epimerase enzyme (UGE) is used at one of the main stages of galactose metabolism in living systems. It causes the conversion of UDP-galactose into UDP-glucose. The gene for this enzyme can be obtained from humans, yeast and bacteria. The present invention contemplates the use of an enzyme encoded by a bacterial gene.

Due to the expression of this foreign enzyme in the plant cell, availability may be reduced

UDF-galactose and the formed useful substance -- UDF-glucose. It can be safely assumed that the expression of UDP-galactose 4-epimerase will lead to a decrease in the biosynthesis of galactinol, which is one of the precursors of non-food sugar glycosides. In addition to reducing the rate of accumulation of unwanted sugar glycosides, the activity of the introduced enzyme can cause increased availability of UDP-glucose and, as a result, the formation of sucrose. The latter is expected to participate and increase the activity of other metabolic pathways that require sucrose, either as a carbon source to increase plant productivity (i.e., for proteins, lipids, total yield, etc.) or directly for sucrose accumulation.

However, in the scope of the present invention, other applications of this method can also be considered. It is possible to i) change the level of various sugar derivatives, such as glucose-1-phosphate and glucose-6-phosphate, using the enzyme phosphoglucomutase (FGM). This enzyme causes the interconversion of glucose-1- and glucose-6-phosphate (Glucose-1-F, Glucose-6-F) in the synthesis and absorption of sucrose. He plays the main role of sou

From the synthesis and use of sucrose, starch and glycogen, it is present in all organisms. The gene for this enzyme can be obtained from a large number of eukaryotes, as well as bacterial sources (for example, Adgobasiegiitis). ise)

Glucose-6-F is the main starting material for a large number of interconversions of sugars, one of which is the synthesis of myosinosit-1-F. The latter is the main substrate and cofactor in the synthesis of phytic acid and, accordingly, non-food sugar glycosides. Expression of the enzyme would, as might be expected, decrease the level of o

Glucose-6-F, which would reduce the level of the aforementioned non-nutritional factors. In this way, within the scope of this invention, it is possible to consider various metabolic changes.

This method is not limited to any particular secondary metabolic pathway. Nor is it limited to any particular type of plant. On the contrary, this method can be used to alter the secondary metabolic pathways common in many economically useful types of agricultural crops, "

Including monocotyledons and dicotyledons, whether to target a secondary metabolite that is unique to a particular crop.

The biochemical basis of the method of the present invention consists in regulating the activity of enzymes due to availability of substrates. In general, the rate of enzymatic transformations is influenced by the availability of substrates.

In other words, the enzyme synthesizes the product in an amount proportional to the amount of available substrate. Reduction

The concentration of the substrate causes a decrease in the formation of the product. In addition, many enzymes are inhibited by the -I end product, that is, the rate of enzymatic transformations decreases in the presence of a large excess of the end product. Therefore, by changing the level of available substrates or enzymatic products, it is possible to control the overall formation of compounds formed by biochemical pathways. c Examples of secondary metabolic pathways that can be changed according to the present invention are the biosynthesis of isoprenoids, the biosynthesis of alkaloids, the biosynthesis of terpenoids, the biosynthesis of phenols, the biosynthesis of sugars

Me, alcohols or any other secondary metabolic pathway in which compounds are formed with non-food or

It has economically useful properties. Specific secondary metabolites that can be modified according to the present invention include non-food phenolic compounds such as sinapine or glucosinolates in cruciferous plants, products of sugar alcohols such as phytic acid or stachyose and raffinose, gossypol in cotton, nicotine, xporogenic acid, condensed tannins or any other non-food secondary metabolite. Chlorogenic acid is commonly found in soybeans, cotton, and sunflower and is formed from caffeic acid, a compound that

F) is formed within the phenylpropanoid pathway. However, other non-food compounds include saponins -- non-food compounds found in many plants, including alfalfa. Saponins are high-molecular glycosides consisting of a sugar component attached to a triterpene or steroid or aglycone. There are at least three classes of known saponins: triterpene glycosides, steroid glycosides, and steroid alkaloid glycosides. The biosynthesis of saponins in plants is based on the starting material - squalene. Other important classes of secondary metabolites such as phytosterols, cardenolides, cucurbitacins, quassinoids and limonides are also formed from squalene. An excess of saponin in the diet of animals is associated with a condition known as bloat. 65 The present invention shows the application of the method in a large number of independent secondary metabolic pathways. However, the applicability of the method of modifying any secondary metabolic pathway in plants will be apparent to the skilled person, and a general method of implementing the invention is proposed.

In one embodiment, the present invention provides methods and DNA compositions for altering the level of sinapin and related phenolic compounds in plants. In another embodiment, the invention provides plant cells with an altered level of phenolic substances and plant seeds with a reduced level of phenolic substances, in particular cruciferous plants with a reduced level of sinapin. In another embodiment, this invention provides methods and DNA compositions for changing the level of phytic acid (a product of the secondary metabolic pathway of sugar alcohols) in plant cells. In another embodiment, the invention provides plant cells with reduced levels of phytic acid and plant seeds with reduced levels of phytic acid. In another embodiment, the invention provides plant seeds with a reduced level of 7/0 phytic acid, suitable for food use, plant seeds with a reduced level of phytic acid, suitable for modified flour, and plant cells with altered sugar alcohol (inositol) metabolism.

Each secondary metabolic pathway in plants has a limited number of associated enzymes and substrates for them, which are themselves unique or used in a unique way. The secondary metabolic pathway can produce various compounds that are used or present throughout the plant. That is, as a means of changing secondary metabolism, unique enzymes are used to specifically change the flow of compounds through a certain pathway. To illustrate the use of the method, two different secondary metabolic pathways were altered.

Below is information about these processes to help you fully understand the nature of this method. (A) Secondary metabolic pathway of phenylpropanoids

Plants synthesize a large number of phenolic compounds through the phenylpropanoid pathway, which is a secondary metabolic pathway. Phenylpropanoid metabolism is involved in many plant physiological processes, including disease resistance, UV protection, and plant growth regulation. The products of this process are used in the biosynthesis of lignin and suberin, which are components of plant tissues and participate in the formation of plant fibers -- a general term that refers to the poorly digestible carbohydrates of grain or flour. Lignin is believed to be involved in the formation of insoluble fibers, so a high level of lignin in grain or flour is associated with inefficient use of grain or flour by monogastric animals. Therefore, plant phenols, especially the phenolic precursors of lignin, can be considered as non-nutritional factors for animal feed. Reducing or appropriately altering the level of plant phenols, particularly phenols used in lignin formation, can produce higher grade forage. Plant phenols are also involved in the formation of various compounds, some of which are also non-food in nature. X,

The invention offers a means of reducing the level of certain phenolic compounds, which are considered non-food, in plant cells, seeds or flour used for feed. In particular, this invention envisages a reduction in the level of the bitter non-food compound sinapine (or sinapoylcholine) in plant cells from which it is synthesized. Sinapin is also believed to combine with protein in feed, reducing the suitability of food protein for assimilation and use by the animal. A bitter taste can also affect consumption levels. In addition, when feeding some breeds of chickens that lay eggs with a brown shell, sinapin causes an unacceptable fishy smell in the eggs. The amount of sinapin in the feed in these cases should be kept below 1095 and this limits the use of sinapin-containing feed. Therefore, the reduction of sinapin in grain or its flour in cruciferous plants is an important economic task. in c The formation of sinapin occurs as an increase in the phenylpropanoid pathway. The pathway defined in this invention includes biochemical pathways leading to the formation of sinapinic acid, as well as biochemical; processes branching from different steps, such as side pathways leading to the formation of lignin monomers and other biochemical substances derived from the products of the phenylpropanoid pathway.

In the phenylpropanoid pathway, I-phenylalanine, an aromatic amino acid, is a substrate of the enzyme I-phenylalanine ammonylase (FAL). The enzymatic activity of FAL leads to the formation of cinnamic acid, which is a substrate of cinnamate-4-hydroxylase (C4H). The product of TsAG is p-coumaric acid, which is the precursor of many o-flavonoid compounds, and some of them can also be formed from I-tyrosine by the action of the enzyme c tyrosine ammonium lyase (TAL). Coumaric acid can also be a precursor of lignin biosynthesis. The enzyme 5or p-coumarate-3-hydroxylase (KHG) acts on p-coumaric acid and forms caffeic acid. Caffeic acid

Me is converted by caffeate/5-hydroxyferulate-O-methyltransferase (OMT) into ferulic acid. Ferulova

Heic acid is one of the three known main phenolic monomers involved in lignin biosynthesis.

Ferulic acid can also be a substrate of the enzyme ferulate-5-hydroxylase (F5H), which forms 5-hydroxyferulic acid, which, in turn, can be converted by the enzyme OMT to sinapinic acid. dv Sbinapic acid is the second main phenolic monomer of lignin. Sinapinic acid can also be conjugated in the form of sinapoylglucose due to the action of the enzyme UDfF-glucose-sinapoyltransferase (UGST).

F) Sinapoylglucose is a substrate for sinapoylglucose: sinapoylcholine transferase (SHT), which leads to the formation of sinapoylcholine or sinapine. The last two stages are widespread in cruciferous plants; in Vgazzisaz, seed-accumulated sinapin represents the main non-polymeric phenolic compound found in ripe seeds. The generalized process of phenylpropanoid metabolism is shown in Fig.2. It should be noted that in some plant species, additional enzymes may be part of the phenylpropanoid pathway.

Sinapine or sinapoylcholine is the most common phenolic compound in cruciferous seeds; in V. pariz, it can add up to 495 flour |Viaiig apa Keiispegi, 1984, 9. Road Adgis. 35:29). Sinapin synthesis occurs in the unripe seed, which is still light green in appearance and is believed not to undergo 65 degradation during ripening. AI., 1993., Agsp. Viospet. Viorpuvz. 300: 622.

Seed germination (ie, seed germination) reduces sinapin levels through the action of esterase, which breaks it down into sinapic acid and choline. It has been suggested, but not proven, that the breakdown of sinapin may provide choline for phospholipid synthesis during seed germination |Zygask et al., 1981., 7.

Maiypogesi. Zbs: 215). However, since most non-cruciferous plants do not contain sinapin in their seeds,

It is unlikely that sinapin is the primary compound for seed germination or seed germination in general.

Mutants with a defective general phenylpropanoid pathway were isolated in Agaridorziz INaiapa. AI,, 1992., Te Riapi Seiyi 4: 1413-1424). These mutants represent excellent material for physiological, biochemical and genetic studies, although they have no agronomic value due to the negative consequences of the mutation, which include increased sensitivity to UV radiation due to the manifestation of the mutant 7/0 phenotype throughout the plant, especially in the leaf. These mutants, designated 5IM1 (sinapoylmalate biosynthesis mutants), block the synthesis of sinapinic acid esters, reducing the level of sinapin (a complex ester of sinapoylcholine) and changing the composition of plant lignin monomers. A change in lignin levels can result in a plant with a unique lignin composition and, therefore, altered fiber.

Sparrie her aiI. consider the use of ZIM1-type mutants to reduce lignin or 7/5 Changes in lignin levels in important cruciferous seed species such as canola, but do not specify the direction in which this could be done. It is also indicated that the reduction of sinapin in rapeseed is an important goal, but it is not determined how this can be done using the 5IM1 mutation. Considering the obvious sensitivity of the ZIMI1 mutant to ultraviolet light, the mutation is not of great importance for use in agricultural conditions. However, although methods of reducing the content of the sinapin component in seeds are not described in the current state of the art, the ZIMI mutation provides important scientific evidence that sinapin is not the main component of plant growth and development, and that seeds with reduced levels of sinapin are capable of germination and development. However, the ZIMI mutation does not provide a way to reduce sinapin levels in agricultural cruciferous grain production without accompanying deleterious UV sensitivity.

In addition to non-food phenolic compounds, such as sinapin, many products of the phenylpropanoid pathway are involved in the formation of lignin. Lignin biosynthesis is a component of the general phenylpropanoid pathway, in which at least three basic phenolic precursors are formed - coumaric, i) ferulic and sinapic acids, the products of which are polymerized into lignin and other phenolic compounds.

The biochemistry of lignin formation from these precursors is complex; there are many other enzymes involved in this process, for example, caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), caffeoyl-CoA reductase (CCOLOMT), cinnamyl alcohol dehydrogenase (DHC), cinnamoyl-CoA- reductase (CKR), peroxidase, 4-coumarate:CoA-ligase (4AKL) and coniferin-specific p-glucuronidase (KBG). Other enzymes may be involved, and the biochemistry of lignin formation is not fully understood yet. yu

Lignin is a complex polymer mainly composed of elements of monomeric phenolic compounds in different proportions and with different types of bonds in different cell types and in different plant species. Lignins are essential components of the plant cell wall, in which tissues undergo mechanical stress or participate in water transport. In addition, lignins are believed to be involved in pathogen resistance mechanisms. In dicotyledons, guaiacyl-syringillignin is usually found, composed of both guaiacyl elements, ferulic acid derivatives, and syringyl residues, sinapic acid derivatives. Thus, both sinapic and ferulic acids are required for the formation of lignin of arbitrary type. The ability to decompose or process wood, for example, when cooking pulp, is believed to be largely dependent on the composition of lignin monomers, which is based primarily on the availability of individual lignin monomers. It is assumed that the presence of a 5-O-methyl group in sinapoyl-derived syringyl lignins reduces cross-linking, which makes lignin composed primarily of syringyl elements easier to process than lignin composed primarily of guaiacyl elements derived from ferulic acid. Thus, methods of increasing the level of syringillignin can lead to the creation of more easily digestible feed due to the reduction of lignin cross-linking.

Digestibility of grain forage crops is affected by the amount of fiber, because cross-linked lignins are resistant to decomposition and physically connect the components of the cell wall. "sl From the above, it is obvious that any attempts to improve grain feed by reducing or eliminating sinapin and changing the total phenolic level should be aimed at seeds that

Pho sprouts. In order to change the biochemical process of sinapin synthesis in the seed itself, the molecular method is the most suitable

HY6) genetic approach, taking into account the lack of endosperm, which naturally provides a low level of sinapin.

In one embodiment, the present invention describes a method that changes the level of phenolic compounds in the seed and also changes the level of sinapic acid and related phenolic compounds. The method is based on the introduction of new enzymes affecting the availability of compounds used as precursors and substrates for enzymes in the phenylpropanoid pathway. Depletion or alteration of a sufficient quantity of these compounds (iF) causes a biochemical alteration of the normal process and the formation of another quantity of various phenylpropanoid products. ko An example of the application of the method is a new enzyme, choline oxidase (HO), which is used to reduce the amount of choline in a plant cell, especially in seeds. Choline is used, in particular, for obtaining sinapine from sinapoylglucose. Decreasing the amount of choline causes a change in the level of sinapine precursors (choline and sinapoylglucose) and therefore changes the composition of precursors formed in the previous enzymatic steps of the phenylpropanoid pathway, including sinapic acid. The result is a seed with greatly reduced sinapin levels and altered phenolic content. It is known that when choline is oxidized by choline oxidase, hydrogen peroxide is formed. The formation of hydrogen peroxide in plant cells is considered beneficial, and therefore there is an additional b5 advantage of the action of choline oxidases on plants. As an illustration of an additional aspect of the method, a second enzyme, betaine aldehyde dehydrogenase (BADH), is used to increase the conversion of the choline oxidase product,

betainaldehyde, to glycine betaine, which is used as a stress reliever. Complex betaine is useful as a compound for various applications in the food industry and as a plant growth enhancer.

Thus, the beneficial effect of changing the phenolic level in plant cells and, in particular, the reduction of sinapin level is evident, due to the reduction of the only precursor in the phenylpropanoid pathway. It will be clear to one skilled in the art that other enzymes may be used within the scope of the method to modify various other end products in the phenylpropanoid pathway.

Another example within the scope of the present invention is the use of a new enzyme for the decomposition of 7/0 ferulic acid, which is also formed in the phenylpropanoid pathway. Among the three main lignin monomers, ferulic acid is believed to provide mechanical stiffness and strength to cell walls through cross-linked pentose chains, arabinoxylans, and hemicelluloses, making cell walls more rigid and less susceptible to enzymatic degradation. Thus, as a component of lignin, ferulic acid plays an important role in the mechanical strength of plant tissues. Decreasing ferulic acid levels appropriately can have many beneficial effects.

Ferulic acid is synthesized within the general phenylpropanoid pathway as described above and serves as a precursor for various products of the process |see URM Kozaglga ei. aiYi., doigpa! oi Ipaivigiai Misgtobiodou 15: 457-471, 1995); (K. MUPeyep apa K. Zedegoyi, Riapi Se 7: 1001-1013, 1995); (KA Oihop apa Mi. Raima, 1995,

Riapi Sei! 7: 1085-1097, 1995).

The presented method provides a means of changing the level of ferulic acid and, due to this change, the production of various other compounds in the phenylpropanoid pathway, in particular sinapine. One aspect of the present invention describes a method by which the level of ferulic acid in plant cells varies. The method is based on foreign enzymes that break down ferulic acid. Some enzymes can also produce compounds that are used economically. For example, the enzyme decarboxylase of ferulic acid is used, which produces the compound vinyltviacol, which can be used as an industrial raw material and can accumulate in plant cells without harmful effects. Depletion or alteration of sufficient ferulic acid i) causes a biochemical alteration of the normal phenylpropanoid pathway and the formation of a different number of different phenylpropanoid products.

The method according to the invention is not limited to the phenylpropanoid process. As an example of the application of this part of the invention, the modification of the secondary metabolic pathway in which sugar alcohols are formed is given. (B) Secondary metabolic pathway of sugar alcohols ise)

The present invention also provides methods and DNA compositions for altering the level of sugar alcohols in plant cells. As a result of the application of the method according to the present invention, the level of compounds derived from sugar alcohols, such as phytic acid, stachyose, raffinose, sucrosylglycosides, uronides and pentoses, phosphoinositides and glycophosphoceramides, changes in plant cells. As a result, in some implementations of the invention, seeds with a reduced level of phytic acid are obtained. The invention also provides seeds with a reduced level of phytic acid, suitable for use in feed, seeds with a reduced level of phytic acid, suitable for the production of modified flour and plant cells with an altered mechanism of inositol metabolism. Also, in this way, the level of other compounds derived from the metabolism of sugar alcohols, such as stachyose and raffinose, changes. Seeds with altered phytate levels are particularly useful for use in feed. z c Phytate is an important component of many types of seeds, typically 2--495 of the weight of the seed, but can reach the level of 1095 in some species. A high level of phytate in the forage diet is associated with loss of appetite, ;" reduced size of offspring and other negative factors. These effects may be due to phytate's ability to bind zinc. Complexes of phytic acid with other seed components are generally referred to as phytin. High levels of phytin are also associated with negative effects. -I In addition to the harmful effect on feed efficiency, the presence of phytic acid in the feed ration also causes a large number of undesirable consequences in the environment. In monogastric animals, the phosphorus bound to phytic acid is generally unavailable, so phosphorus must be added to the ration, which causes additional c costs. Phosphorus bound to phytic acid is excreted by monogastric animals, and subsequent decomposition of feces by microorganisms leads to the release of phosphorus into the environment, which

Me, contained in phytic acid. The high level of phytic acid in many grain feeds causes significant

He is the amount of phosphorus released. The long-term increase in livestock has often caused the overgrowth of water bodies and other environmental problems related to phosphorus pollution. As expected, these problems will become more significant and may become the main limitation for increasing the number of livestock in the future. Thus, methods of reducing the level of phytic acid release will significantly affect the solution of environmental problems.

F) Although the actual costs associated with the addition of phosphorus to the diet do not constitute a significant part of the cost of feed, the consequences for the environment due to the phosphorus pollution released cause significant additional costs that could be avoided by producing flour with a low level phytate In ruminants, the presence of microbial fauna in the rumen can cause the release of phosphorus contained in phytic acid, making phosphorus more available. As a result, the amount of added phosphorus in the diet is significantly reduced compared to monogastric animals. However, the amount of phytic acid in most plant seeds exceeds what is actually needed, so even in ruminants, phosphorus pollution is a major problem. 65 So far, the main methods of reducing phytate have largely been aimed at the decomposition of phytate by the action of phytase enzymes, which are part of the feed. Although this method leads to greater availability of phosphorus, it is not suitable for the production of grain or feed with reduced levels of phytic acid, but only for feed in which the phosphorus contained in phytic acid is generally more available. Thus, the method using the enzyme phytase is used only to make phosphorus in phytic acid more available.

Phytase activity is usually observed in such microorganisms as molds (Azregaiiiv), bacteria (Vasiiv5, Rzepdotopaz) and yeast (Zasspagotusev). The removal of phytates by these plant materials using enzyme mixtures containing microbial phytase is described, for example, in US Pat. No. 5,554,399,

Most microorganisms synthesize a large number of varieties of phytase, which may include phytase itself along with various phosphatases that further break down phytic acid. The complete release of available phosphorus from 7/0 phytic acid may depend on a number of different enzyme actions.

An alternative variant of plant transformation for the production of microbial phytase is also described. In the patent

USA Mo5593963 describes the action of phytase in transgenic plants under the control of regulatory sequences, which are able to direct the expression of the enzyme either constantly, or specifically to the stage of the process or tissue-specifically. Plant cells or, more specifically, plant seed cells can be created that /5 Contain the enzyme phytase, which can release some of the phosphorus contained in phytic acid.

However, simply releasing phosphorus from phytic acid does not fully address the problem of reducing phosphorus waste and the potential non-nutritional effects of phytic acid. The more important presence of complex phytic acid or phytate is not reduced. Means for releasing phosphorus from phytic acid are known in the literature, but there is no means of controlling the level of phytic acid produced.

Any means by which phytic acid levels can be conveniently controlled would be very useful in feed production. For example, plant seeds with reduced levels of phytic acid would make it possible to create feed with more digestible minerals. The phytic acid-reduced seeds would also produce a phytin-reduced grain feed that would be more nutritious and easily digestible due to the reduced phytic acid complexes. high school

In addition to flour with increased value, flour with reduced levels of phytic acid would result in less phosphorus entering the environment and less pollution. Although means of releasing i) phosphorus from phytic acid can reduce the level of added and released phosphorus, the actual level of phytic acid and therefore potentially available phosphorus exceeds the dietary phosphorus requirements of most animals. Thus, methods of controlling the level of phytic acid will find use in the application of feeds and, in addition, will allow the creation of new and useful flour and food compositions. ise)

Given these circumstances, it is clear that a means to control phytate production during seed germination could be useful and address the problems associated with phosphorus pollution and non-nutritive effects of phytic acid. In addition, a genetic mechanism that could be applied across a wide range of plant species would be particularly useful. The presented invention provides (M such solutions.

An understanding of the biochemical mechanism responsible for the formation of phytic acid is required to assess the capabilities of the present invention. Although the biosynthesis of phytic acid is not completely understood, a large number of key steps are known. Phytic acid is a hexaphosphate derivative of myoinositol; the biochemical process that synthesizes phytic acid uses myoinositol, a sugar alcohol, exclusively as an initial substrate. This compound (myoinositol, also commonly referred to as inositol) is also key in the production of . other derivatives and epimers of myoinositol. Some of these derivatives, such as sucrose glycosides, are also non-nutritive compounds, so reducing their levels is also desirable.

Myoinositol is a sugar alcohol found everywhere in plant cells. However, simple protection

Each of the hydroxyl groups of myoinosit makes it unsuitable for the biosynthesis of phytic acid and other -I processes. Protection of myoinosit can be carried out directly by methylating individual sites with various methyl-transferases. For example, myoinositol is converted to ononitol by monomethylation at position o b. Methylation at position 5 produces sequoitol, which epimerizes to pinitol. Pinitol cannot be used for the biosynthesis of phytic acid. Methylated derivatives of myoinositol are known to confer beneficial properties to the plant, such as stress resistance, and participate in the transport of solutes and

Me, stabilization of membrane proteins. Thus, the modification of inositol with the help of non-specific enzymatic

Of the actions, usually not related to the metabolism of sugar alcohols, is considered within the scope of the present invention.

The biochemistry of phytate formation is not yet fully understood, but at least two potential formation processes are known, and there are likely a large number of different enzymes involved in the biosynthesis of the two phytic acids. Phytate is found in most plant tissues, but is especially abundant in seeds and pollen, playing a predicted role in phosphorus conservation. Therefore, it is difficult to invent a means to change the biosynthesis of phytate

F) in the usual way, in particular for the reason that the biochemistry of its formation has not yet been clarified. In order to minimize the production of phytic acid, the present invention describes methods and DNA compositions encoding enzyme(s) that modify myoinositol, preventing its use in phytic acid biosynthesis. because this invention envisages the use of a foreign methyltransferase gene for the selective methylation of myoinositol in seeds, in particular in seed tissues responsible for the biosynthesis of phytic acid (foreign means an enzyme not usually associated with phytate biosynthesis in this plant cell). The invention uses a foreign enzyme obtained from a halophyte plant that is not found in common agricultural plants, such as cereals, soybeans, cotton, alfalfa, wheat, barley, rye, sorghum, 65 Sunflower, Vgazzis oil crops and other traditionally grown crops.

The production of methyl inositol from mysinosite also has the added benefit of producing a harmless compound with no harmful effect on the plant cell. Thus, due to the reduction in the amount of available myoinositol, the production of phytic acid is reduced.

Phytate is one of the main non-nutritive factors in grain feed. The non-nutritive effects of phytic acid include binding of minerals, formation of complex compounds with protein and other negative effects, in particular, associated with the release of excess phosphorus in the form of phytic acid, which is transformed in the environment, causing phosphorus pollution. Thus, methods of reducing the level of phytic acid in plant cells are used for feed. In aquaculture, high levels of phytic acid are also a major problem when using plant protein as a fishmeal substitute. Therefore, 70 ways to reduce the level of phytic acid in plant cells, especially in the cells of tissues used for feed, are useful and may find wide application. Methods for reducing phytic acid levels in seeds of a wide range of forage plant species are particularly useful for the feed industry.

The plant gene encoding myoinositol O-methyl-transferase (IMT) was isolated from Mesetrguapipetit 7/5 bguziaipt (crystal grass) and this enzyme was shown to convert myoinositol to pinitol in heterologous transgenic plants |US Patent Mo 5,563,324 ). This plant gene was put under the control of a constitutive promoter to increase plant stress tolerance by overproducing the methylated derivative of myoinositol, ononitol, which can subsequently epimerize to pinitol. In the present invention, the plant gene is under the control of a seed-selective promoter to alter inositol metabolism in the seed.

Production of pinitol, described in US patent No. 5,563,324, was shown to increase salt tolerance when constitutively applied to transgenic tobacco. Similarly, the constitutive effect of bacterial 1-P. of mannitol dehydrase to the plant cell causes the formation of mannitol, a sugar alcohol, or polyol, and confers salt tolerance on the cell. Thus, US patent No. 5,563,324 describes the production of sugar alcohols using an enzyme capable of producing sugar alcohols from sugars inherent in plant cells as a means of imparting salt tolerance to plant cells.

However, US patent Mo5563324 does not contain any suggestions for using a gene capable of modifying i) myoinositol to prevent its use as a precursor for other compounds including its own derivatives. However, US patent Mo5563324 clearly shows that the modification of myoinositol does not lead to any negative effects for the plant, and therefore manipulation of the level of myoinositol is not expected to lead to any harmful effects.

Accordingly, the possibility of modifying myoinositol with a methyl-transferase gene will probably not be harmful to plant cells, and the methylated product of this enzymatic reaction is known not to be harmful to plant cells.

However, myoinositol is key in many different aspects of plant growth and development. In addition to its role as a precursor for the biosynthesis of phytic acid, myoinositol is also used for the biosynthesis of pentose and uronides, and it is also present in the phosphoinositides of plant cell membranes, as well as in other complex lipids of plants, including glycophosphoceramides. In addition, it is also a precursor to other natural isomers of inositol, with many of these, as well as myoinositol, distributed as methyl esters in a species-specific manner throughout the plant world. "

The role of myoinositol in the general metabolism of plants is significant, and modification of the level of myoinositol can have unforeseen consequences, especially in agronomic conditions. Although US patent Mo5563324 describes constitutive action of the methyl-transferase gene, it does not provide any evidence that the resulting plants will be useful US patent Mo5563324 states: "Even if the newly inserted genes do not cause the plant to perform better under agricultural conditions, transgenic plants containing such genes will be used to study how changes in internal plant processes (eg, osmoregulation)-I will affect yield plants." (column 2, lines 60-65). Therefore, US Patent No. 5,563,324 does not provide for control of myoinositol levels to reduce phytic acid levels, and it is not necessarily expected that the research described therein will result in plants with beneficial agronomic characteristics. c Also, US patent Mo5563324 describes the action of a gene modifying myoinosit in the whole plant, with the aim of increasing resistance to stress or for further scientific research.

Me. Thus, it can be confidently stated that the present invention offers a completely new one

Ge approach to reducing the level of phytic acid. The use of the methyl transferase gene is specific for reducing the level of phytic acid. The present invention does not apply to already known methods, such as the action of phytase enzymes, which do not solve the problem of excess phytic acid in seeds and/or grain flour. In the course of the work done, it was established that the level of phytic acid can be controlled by changing the level of myoinositol available for phytic acid biosynthesis. It was found that the action of the methyl-transferase gene on

F) seed tissue leads to a decrease in the level of phytic acid in seeds without any noticeable changes in other characteristics of the plant. It is obvious that limiting the expression of the methyltransferase gene in seed tissues responsible for phytate biosynthesis significantly reduces the level of phytic acid in ripe seeds without any additional effects on the plant, even grown in extreme field conditions. The use of a tissue-selective promoter offers many advantages over known methods, including limiting enzyme action to seed tissue while preserving myoinositol metabolism in other tissues.

The purpose of the present invention is to prevent the use of myoinositol as a starting material for the biosynthesis of phytic acid by transferring it to a metabolic process in which it is transformed into compounds harmless to the plant. This method is based on novel enzyme actions, including, in one embodiment, a methyltransferase gene that depletes or alters the amount of myoinositol used to produce phytic acid. Depletion or alteration of the level of this precursor causes a biochemical alteration of the normal process of phytic acid biosynthesis and a decrease in the amount of phytic acid produced.

The biochemical basis of the present invention is based on the concept of regulation of enzyme activity due to the availability of substrates. In general, the efficiency of enzymes is affected by the availability of substrates. In other words, the enzyme produces a product in an amount proportional to the amount of available substrate. A decrease in substrate concentration causes a slower rate of product formation.

Thus, the present invention takes advantage of this approach by using a new enzyme, methyltransferase, which depletes the availability of inositol, the substrate used for 7/0 Formation of phytic acid. Any enzymes capable of affecting the amount of myoinositol in plant seeds can be used within the scope of the present invention. The method is based on the action of an enzyme that in any number of cases changes the level of myoinositol used for the biosynthesis of phytic acid.

In contrast to the US patent Mo5563324, the presented invention involves the modification of phytate production in a seed-selective manner by the action of a gene capable of modifying myoinosit, making it unsuitable for the production of phytic acid. Surprisingly, even uncontrolled action of the gene that changes myoinosit causes a decrease in the level of phytate in the seed. However, in the most preferred embodiment of the present invention, a seed-selective promoter is used to ensure that the agronomic performance of the plant is not compromised and the resulting plant is normal in all respects except for reduced phytate levels in the seed. Therefore, in contrast to the US patent Mo5563324, the presented invention does not provide for obtaining either plants with increased resistance to stress, or plants for research purposes.

The described method offers many advantages over known methods associated with reducing the level of phytic acid due to the action of phytase enzymes. These include plants with altered levels of phytic acid in individual tissues, plants with seeds with reduced levels of phytic acid, and grain feed with reduced levels of phytic acid. The advantages of such plants, seeds and flour include better feed utilization and feed efficiency, production of flour and feed with reduced levels of phosphorus and therefore better environmental performance, and the ability to be used in the feed industry, which was previously i) limited by the amount of non-food phytin acids in flour or feed.

The method described is useful for many types of plants, including monocotyledonous and dicotyledonous plants, as well as both grain and oilseed crops. It is particularly useful for oilseeds and cruciferous plants.

The result of this genetic modification is plant cells with altered metabolism of sugar alcohols, particularly inositol metabolism, and plant cells with a reduced level of phytic acid, in particular seeds with a reduced level of phytate. Thus, the method of the present invention offers a useful means of altering the secondary metabolism of sugar alcohols. at

Although an embodiment of the present invention that causes a change in the metabolism of sugar alcohols, for example, plant cells with a reduced level of phytic acid, has been demonstrated in rapeseed plant cells, grain plants also have a high concentration of phytic acid and a reduction in the level of phytic acid in grain cells can be achieved similarly. by.

The present invention provides methods, genetic constructs and vectors used to "

Modifications of phytate levels in plant cells and seeds, especially to reduce phytate levels in plant cells. The invention also offers methods and DNA compositions used to reduce the level of phytate in . seed plants. In addition, the invention relates to modified seed meal and animal feed, what? contains modified flour, especially flour with a reduced level of phytate.

Reducing the level of phytic acid is carried out using recently described methods and DNA mixtures.

The method involves the development of a "metabolic bypass" or a new biochemical process capable of -I diverting a precursor in the process of phytic acid biosynthesis to a new and newly introduced "dead end" that reduces the production of phytic acid. As a result of one implementation of this method, "dead-end pathway" 1 produces compounds that are known to be harmless to this plant cell. Thus, it is safe to say that the bypass process of converting myoinositol precursor compounds into these compounds is not a threat

Life activities of plants.

According to another embodiment of the invention, recombinant DNA vectors and constructs are provided for the creation

He plant seeds with an altered level of phytic acid, in particular plant seeds with a reduced level of phytic acid. In addition, another embodiment of the invention provides a plant seed useful for the production of grain flour with a reduced level of phytate. This flour contains a reduced level of phytate and is therefore useful for animal use, especially for those industries where the level of phytic acid is associated with environmental pollution or identified as a non-nutritive factor. The invention also offers (F), a modified flour obtained from genetically modified seeds, which can be used in various animal diets, including for poultry, pigs, cattle and fish.

The method includes the addition of a new enzyme that can change the level of myoinositol in seeds and can be directly applied to all types and varieties of plants. The same gene can be used for any type of plant because the myoinositol change will occur in the same way in any plant. The method is not based on the use of gene suppression technologies, which depend on the DNA sequence and therefore require specifically adapted genes for each individual gene or plant species.

Thus, the application of the presented invention to all cultures is obvious. Numerous suitable enzymes of microbial, plant or animal origin can be used within the framework of the method. The same genetic constructs can be used in different plant species.

Thus, in view of the broad aspect of the present invention, there is provided a method for reducing phytate levels in plant seeds, comprising the construction of a plant transformation vector which, in addition to a switchable marker to identify plant cells transformed by said vector, contains an enzyme capable of metabolizing myoinositol, which makes it unsuitable for the formation of phytic acid.

In the context of the present invention, inositol metabolism includes hydroxylation, isomerization, methylation, cleavage, or any other biochemical modification that renders myoinositol insensitive to the action of the appropriate enzymes that normally act on myoinositol to produce phytic acid. Therefore, 70 metabolism includes any modification that effectively reduces the availability of myoinositol from the set of available substrates for phytic acid biosynthesis.

As an example of the presented invention, the gene encoding myoinositol methyl transferase is used. This enzyme, under the control of a promoter selective for action in plant seed cells, is capable of depleting the amount of myoinositol available for the production of phytate in this seed. In another aspect of the present invention, this methyltransferase is capable of converting myoinositol into a harmless substance.

Therefore, in general, plant cells created by the method according to the present invention retain an unchanged basic metabolism and have phenotypes that do not differ from unmodified plants, except for the change of the product or products in a separate process of secondary metabolism.

In order to ensure that the primary metabolism remains unchanged, the present invention describes a tissue-specific method for the formation of a secondary metabolite by altering the availability of a substrate specific to the secondary metabolic process and important for the formation of the end product of the process, in particular substrates in one-p' biochemical stages of formation of the final product. Thanks to this, a reduction in the number of individual products in the process of secondary metabolism is achieved.

In the context of the present invention, compounds used as precursors of the secondary metabolites of phenylpropanoid metabolism include, but are not limited to, cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid, sinapic acid, choline, various i) sinapoyl compounds including, but not limited to, sinapoyl glucose and sinapoyl malate, and sinapoyl choline. In the context of the present invention, the products of the phenylpropanoid pathway include, but are not limited to, flavonoids, lignins, various monomeric and polymerized phenolic compounds, and sinapin. By choosing a specific enzyme, any number of changes in the level of products in the phenylpropanoid pathway can be achieved. Enzymes used to modify precursor compounds can be of microbial, animal (ie) or plant origin. Enzymes can be natural or obtained by mutating known enzymes to change their specific action on a substrate, and thus create a new enzyme.

Enzymes used in the present invention may be capable of modifying compounds that are used as precursors of products formed in the phenylpropanoid pathway by isomerization, conjugation, phosphorylation, hydroxylation, oxidation, dehydrogenation, methylation, or any other similar process. biochemical operation (including binding or sequestration), or may include enzymes capable of degrading compounds used as precursors in the phenylpropanoid pathway, such as hydrolases, decarboxylases, oxidases, esterases, or any other enzymes capable of degrading "Cinnamon acid, p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid, Se) with sinapic acid, sinapoyl-glucose, sinapoyl-malate or choline (used to form sinapine from sinapoyl-glucose). One aspect of the method uses enzymes that produce substances that protect against stress from compounds used as precursors to common products of the phenylpropanoid pathway. Similarly, an enzyme for the specific reduction of an additional substrate that is not produced as part of the phenylpropanoid pathway, but is required for the formation of product -I of the phenylpropanoid pathway, can be used to reduce the level of this product. Accordingly, both compounds used as precursors in the phenylpropanoid pathway and any other compound required in the phenylpropanoid pathway to form products can be considered within the scope of the presented method.

In the context of the present invention, to compounds used as precursors of secondary products

Me, the process of metabolism of sugar alcohols, include, but are not limited to, myoinositol, galactinol, and UDP-derivatives

Ge of sugar. Metabolic products of sugar alcohols include, but are not limited to, phytic acid, stachyose, raffinose, sucrosyl glycosides, uronides and pentoses, phosphoinositides, and glycophosphoceramides. In the context of the present invention, enzymes used to modify compounds of sugar alcohol metabolism may be capable of hydroxylation, isomerization, methylation, cleavage, or any other biochemical modification (such as binding or sequestration) that renders the sugar alcohol insensitive to influence (F) of the corresponding enzymes that usually act on it. In the example of the presented invention, methylation of inositol, which is a precursor of phytic acid, in order to reduce the availability of inositol for the biosynthesis of phytic acid is shown as part of the secondary process of metabolism of sugar alcohols. Thus, it is clear to a person skilled in the art that the precursors of the final product in the process of secondary metabolism are those biochemical substances that are used directly for the formation of a secondary metabolite, or those biochemical substances that are used in a biochemical reaction that leads to the formation of a separate secondary metabolite, and that these biochemical substances are not the main metabolites.

Enzymes acting on compounds that function as precursors of products in the secondary metabolic 65 process preferably form from the aforementioned compounds substances that are safe for the plant cell and therefore can accumulate to a high level without changing the properties of the plant cell. Enzymes can also form substances that have a beneficial effect on the plant cell, for example, they produce substances that protect against stress from these precursor compounds of products of secondary metabolism. The enzymes used can also form substances that are used, for example, as industrial raw materials, pharmaceutical or medical-food substances. Within the framework of the presented invention, one or more enzymes can be used and several different properties can be used to reduce the level of individual compounds-precursors of the product in the process of secondary metabolism.

Enzymes used in the context of the present invention can be obtained from many sources or by many methods. For example, it is possible to identify a precursor compound in the 7/0 process of secondary metabolism to which the present invention should be directed. The chemical structure of these compounds is well known or can be determined, so the enzyme literature can be analyzed to identify an enzyme with known activity on chemically similar substrates. Accordingly, a suitable enzyme and gene can be identified and used in the context of the present invention. In addition, combinatorial chemistry or similar schemes that search for artificial proteins with the desired /5 property can be used to obtain a synthetic gene. Enzymes used in the framework of the present invention can be modified using methods generally accepted in this field.

As an example of enzyme modification, a gene encoding an enzyme capable of acting on chemically similar compounds used as precursors in the process of secondary metabolism can be modified and/or selected for increased activity using methods such as site-specific modification, mutation, and selection of altered features in nonspecific systems, such as bacterial or fungal cells. This may include expression of the gene encoding this altered enzyme in bacterial cells to render it incapable of converting the above substances, selection of bacteria producing this enzyme and able to grow on the above compounds using them as the sole nutrient medium or carbon source, and restoring the activity an enzyme capable of acting on a separate compound-precursor in the process of secondary metabolism. Thus, it is possible to create an enzyme with a specific action on a single compound used as a precursor in a specific process of secondary metabolism. It is also possible to breed bacteria i) which will grow on various compounds used as precursors in the process of secondary metabolism.

A specialist can easily perform the selection of mutated bacteria or other cells on individual compounds used as precursors in the process of secondary metabolism, and a means of determining the specific enzymes capable of converting these compounds into harmless substances. This approach may involve direct selection of specific enzymes or stepwise modification and selection of enzymes with desired activity. Similarly, the use of known enzymes now in commercial use, for example, enzyme preparations from extracts of Tgispodegta fungi, capable of degrading lignin in pulping operations, may provide a source of suitable enzyme, for example, for the isolation of enzymes involved in the decomposition of Scheo , phenolic compounds. It is obvious to those skilled in the art that genes encoding enzymes used in the pulp and paper industry to process lignin can be used in the method, either directly or after modification to specifically act on one of the precursor compounds in the phenylpropanoid pathway. It is also contemplated that a large number of different enzymes may be used within the framework of the present invention. Thus, the method is not limited by the source or specificity of the enzyme used. "

For example, in one aspect of the method, an enzyme expressed in a plant cell acts on a precursor compound or product substrate in the phenylpropanoid pathway and produces a substance that is no longer affected by the enzyme that normally uses the compound as a precursor in the phenylpropanoid pathway. ;" Accordingly, the level of a separate product of this process decreases due to the reduction of precursors necessary for its formation. The resulting plant cell can be used directly or to regenerate a whole plant with an altered level of phenylpropanoid pathway products, which will be useful in -And any industrial processes, not only for the production of feed, cellulose or the creation of plants with new compositions of phenolic compounds, including excessive production of individual products in the phenylpropanoid pathway. o Trees with altered levels of phenylpropanoid products will be useful in the pulp and paper industry. Plants with altered levels of phenylpropanoid products will be useful in the feed industry.

Me, The method is also based on the recovery and use of plant cells or tissue with changes

With the properties, in particular, of plant tissue used for the manufacture of fodder or in other industrial processes. It is possible that the changes in the phenylpropanoid pathway envisaged within the framework of the present invention may also cause positive changes in the agronomic efficiency of the plants obtained by these

Methods, especially those aspects of the invention, in which the action of added enzymes leads to the formation of substances that protect against stress. Other applied effects can be expected, such as increased resistance

F) to diseases and ultraviolet radiation, mechanical strength, etc. In another embodiment of the invention, the method includes the step of growing the aforementioned plant cell and regenerating the plant with altered products of the phenylpropanoid pathway. 60 In yet another embodiment, the invention includes the use of the aforementioned plant for an industrial process, such as the production of useful compounds, animal feed or cellulose.

Genes encoding enzymes capable of acting on precursor compounds in the phenylropanoid pathway may be natural, synthetic, or a combination thereof. One of ordinary skill in the art will readily appreciate that the coding sequence of a gene can be altered for more efficient action in plant cells. This can be done by changing the 65 codon sequences so that they are more similar to the codon usage of the normal gene action in a plant cell. In addition, it is obvious that individual restriction sites can be defined for convenient manipulation of the coding sequence. It is also contemplated that the addition of various sequences, such as translation enhancers, introns, etc., may be used to ensure appropriate action of the coding sequence. Enzymes can also be modified by changing substrate binding sites or other modification of the initial sequence of amino acids, which increases enzyme activity. All of these manipulations are known in the art and will be readily understood by a person skilled in the art.

In another aspect of the present invention, the enzyme is under the control of a tissue-selective promoter, in particular a seed-selective promoter. A seed-selective promoter is a promoter that restricts the action of sequences exclusively or predominantly to the seed tissues of a plant. Therefore, the level of a single 7/0 product of the phenylpropanoid pathway changes in a tissue-selective manner, while other plant tissues maintain a normal level of products of the phenylpropanoid pathway. The tissue in which the enzyme acts selectively is used in the preparation of fodder or in any other industrial process.

It is obvious to a person skilled in the art that any number of tissue-selective promoters can be used within the scope of the present invention. In particular, a seed-selective promoter is used to alter the 7/5 level of products of the phenylpropanoid pathway in crops whose grain or meal is used for feed. In other cultures, different tissue-selective promoters can be used depending on the part of the plant in which a change in phenolic content is desired. For example, a root- or tuber-selective promoter can be used to alter the level of phenolics or lignin in roots or root crops such as potatoes, turnips, cassava, and the like. In those cases where leaves or stems are used as feed, a 20 promoter selective for leaves or stems can be used to limit the action of the enzymes on the precursor compounds. in the phenylpropanoid pathway. Other tissue-selective promoters can be used, for example, in trees grown for pulp. Therefore, a promoter that limits the action of the enzyme on the wood will be useful in the framework of the presented method.

In one specific aspect of the present invention, the action of the coding sequence of a choline-converting enzyme in plant cells is envisaged. The action of this choline-metabolizing enzyme depletes the amount of available choline used in the formation of sinapine from sinapoyl-glucose. Some species of i) plants, especially of the family Sgysiiegae, produce the non-food compound sinapin. Sinapine is believed to be synthesized by changing the glucose component in sinapoyl-glucose (VIP-dIsh) to choline. Reducing the level of the non-food compound sinapin increases the value of the seed and the feed obtained from it. The use of an enzyme that converts c to choline, such as choline oxidase, is useful in the present invention.

It is known that choline is oxidized to betaine in food plants such as wheat, spinach, sugar beet and x, barley (Kpodez apa Napsop, 1993, Appia! Kemiemzhm oi Riapi Rpuzioiodu apa Riapi Moiiesshiag Viiodu, 44: 357-384). yu

This oxidation process is catalyzed by two enzymes (known in genetics as the "choline oxidation system"); the products of this process are shown below: o

Choline - betaine aldehyde - glycine betaine (betaine) cha

The reaction at the first stage is catalyzed by choline dehydrogenase (CHO) or choline oxidase (HO) in bacteria and animals (KogmjadomuvKki, Kpaspayigiape apa beimaga), 1991, otsgpa! oi Vasiegioiyudu, 173: 472-478) and choline monooxygenase (SMO) in plants (Kpodez apa Napsop, 1993). The reaction at the second stage is catalyzed by betaine aldehyde dehydrogenase (BADH) (KPodez apa Napsop, 1993). Accordingly, the use of a choline-converting enzyme can lead to a reduction in the amount of available choline in a general or specific manner using constitutive, adaptive, or tissue-specific promoters. Reducing the availability of choline reduces the production of sinapin. In addition, a decrease in the level of sinapin can cause changes in the amount; other products of the phenylropanoid pathway, which leads to the formation of plant tissue with a changed phenolic content.

In another specific implementation of the presented invention, the microbial gene choline oxidase (HO) |Kogmadomuzki, -I Kpaspayigiape apa Zeimaga), 1991, Shotsgpa! oi Vasiegioiodu, 173: 472-478| was placed under the control of a seed-selective promoter. The action of choline oxidases in seeds converts choline, one of the precursors in the biosynthesis of sinapin in rapeseed, into a harmless substance betaine, by forming betaine aldehyde, which is slowly transformed into betaine under the action of the XO enzyme. In seeds with recombinant DNA, the level of 5p binapine decreased and the level of other products of the phenylpropanoid pathway changed. There is flour obtained from such seeds

Me. useful for use in feed.

Ge In another specific embodiment of the present invention, the XO gene was used under the control of a seed-selective promoter, and a second enzyme, BADH, was used to increase the formation of betaine from betaine aldehyde. In this implementation |Vaud, G.A. Her. Adat, G.E. Reispeg, A. MesNidpep, K. Nigi, apa ov Z. Zeimaga)., 1990, bepe 103:45-52.| BADH acted under the control of a seed-selective promoter.

Betaine is a compound found in the edible parts of many food and forage plants; it is also (F, used in some cases as a food and feed additive. Betaine also has a stress-protective function. In another specific implementation of the present invention, the XO gene is used in a cell of a cruciferous plant (Vgazzis species) under the control of a seed-selective promoter, and a second enzyme was used for BADH, also under the control of a seed-selective promoter, to ensure the formation of betaine from betaine aldehyde.

A change in the level of choline in cruciferous seeds implies a decrease in the level of sinapin and a subsequent change in the amount of various products of the phenylpropanoid pathway. It is safe to say that a change in the level of choline in the seed leads to an increase in the level of sinapoylglucose or other sinapoyl compounds, such as sinapoyl malate, b5, which in turn changes the level of sinapic acid, a precursor of lignin. Therefore, the mechanism of biochemical regulation is activated, which is usually referred to as end-product inhibition, causing a change in the level of various products in the phenylpropanoid pathway. Accordingly, preventing the formation of sinapin leads to a change in the level of a large number of different products formed in the steps of the phenylpropanoid pathway before the final step of sinapin formation.

The secondary process of phenylpropanoid metabolism can also be affected by an enzyme that acts on ferulic acid. Ferulic acid is another precursor in the phenylpropanoid pathway. Accordingly, the level of ferulic acid decreases. A decrease in the availability of ferulic acid causes a change in the amount of phenolic compounds, including a decrease in the level of sinapin in cruciferous plants.

Genes encoding enzymes capable of affecting ferulic acid can be natural, synthetic 70 or a combination thereof. One of ordinary skill in the art will readily appreciate that the coding sequence of a gene can be altered to function more efficiently in plant cells.

According to another aspect of the invention, the gene encoding an enzyme capable of affecting ferulic acid is placed under the control of a tissue-selective promoter. Therefore, the level of ferulic acid changes in a tissue-selective manner, maintaining a normal level of ferulic acid in other plant tissues. /5 The tissue in which the enzyme acts selectively is used in the preparation of fodder, food or in any other industrial process.

Enzymes contemplated for use in these embodiments of the present invention include enzymes capable of modifying ferulic acid, and particularly those known to produce useful compounds from ferulic acid. It is clear to a specialist that within the framework of the method of metabolizing ferulic acid, it is possible to use other enzymes. Known sources of ferulic acid-metabolizing enzymes are found in such microorganisms as (Aegorasieg zr, Vasii5 teda(egisht, V. ziBiiv5, Soguperasiegisht dishatisit,

Epiehorasieg zr., Rezcedotopaz zr., Zigeriotusevz zr., AvzregaoiYiyiv zr., Sapaida zr., Ryvagisht zr., Napsepsha zr., Repisiysht zr., Kpodoiogtsia zr., Zasspagotusez zr.)|. This list is not exhaustive, but shows many known sources of ferulate-metabolizing enzymes. Thus, this method is not limited to the sources of enzymes used for the metabolism of ferulic acid. Within the framework of the presented invention, it is envisaged to change the gene encoded by microbes to ensure optimal action in plant cells. Such methods are well (8) known in the art.

As an example of such enzymes, ferulic acid decarboxylase from B. rhytii85 is used. The gene for this enzyme was isolated and its sequence studied. (Lado ei. ai., Arrie4y apa Epmigoptepiai! p

Zo Misgtoriyodu 61:4484-4486, 1995). The gene sequence can be changed to be more similar to the plant gene while maintaining the intended amino acid sequence. This enzyme is capable of creating ix, vinyl guaiacol from ferulic acid by decarboxylation. Accordingly, in one example of the method, a useful compound (viniltvayacol, MO) is formed by the activity of the introduced gene, which can affect ferulic acid. The enzyme does not require additional factors and has a probable molecular weight of approximately 22 mol TlH. uh-

In another specific embodiment of the present invention, the enzyme ferulate decarboxylase is placed under the control of a tissue-selective promoter, resulting in the creation of a plant containing tissues with specifically altered metabolism of ferulic acid.

A change in the level of ferulic acid involves a decrease in the level of ferulic acid and a subsequent change in the number of different products of the phenylpropanoid pathway. The present invention also provides for the creation of plant tissue, particularly grain, useful for use in food and feed. Both the direct use of grain and the use of modified flour from the obtained grain are included in the scope of the presented invention. A reduction in the level of phenols in grain is desirable in feed and genetically modified seeds with altered phenolic content, in particular with less

The amount of non-food phenols is very useful in the production of fodder. -I In addition to direct feeding with whole or partially ground grain, there are many ways to prepare grain flour. Of particular importance for cruciferous seeds is the method of making flour by extracting oil from the seeds of oil-bearing cruciferous crops. Generally speaking, oil is obtained from oilseeds with or without a solvent. The cake or pressed solid residue obtained during this process is used for the preparation of feed and usually contains most of

Me, phenolic compounds from cruciferous seeds. The method of production of flour with a lower phenolic level

They create a new composition that has not been found in cruciferous seed flour at this time.

The presented invention involves the use of genetically modified seeds, created according to the method of obtaining a feed product with a changed level of phenols.

The described method provides many advantages in terms of changing the plant's phenotype. In addition, the method allows the production of various compounds with known industrial use. The described method is applicable in (F, any types of plants or plant tissues. The described method offers many advantages compared to the known methods of controlling the phenylpropanoid process. This method is not based on mutations, such as the 5IMI mutation, which is found in the whole plant and because it is related to UV sensitivity. The method does not use genetic mechanisms designed to block gene expression, such as antisense RNA or cosuppression, which require cloning of plant genes from the phenylpropanoid pathway. The method does not alter any genes inherent in the plant. Accordingly, different functions process such as the production of flavonoids, UV protective substances, participation in disease response and other biochemical functions of the process are not harmfully altered.With tissue-selective promoters, phenolic levels can be changed in any tissue while sparing other plant tissues unchanged.

The described method is used for many types of plants, but it is most useful for cruciferous plants. As a result of using the method, there is an additional advantage due to the production of a stress-protective or industrially useful compound, as a result of the action of the added enzyme. It is obvious that the method

Any number of steps in the phenylpropanoid pathway can be targeted, and other biochemical processes can also be altered by a similar approach.

The presented invention also finds application for changing other processes of secondary metabolism.

For example, one embodiment of the present invention provides methods and DNA constructs for creating plants with altered metabolism of sugar alcohols, in particular by changing the level of phytate in seed cells. Phytate 7/0 is formed from the sugar alcohol myoinositol, which is also a precursor or intermediate for the formation of other compounds such as stachyose, raffinose, sucrosyl glycosides, uronides and pentoses, phosphoinositides, and glycophosphoceramides. In addition, the invention provides methods and DNA compositions for reducing the level of phytate in cruciferous seeds. The invention also provides a plant seed with a reduced level of phytic acid suitable for feed applications, a plant seed with a reduced level of phytic acid suitable for the manufacture of 7/5 Modified feed, and plant cells with a modified metabolism of inositol.

The present invention provides methods and DNA constructs for generating plants with altered levels of phytate in seed cells. The invention also offers methods and DNA compositions for reducing the level of phytate in the seeds of monocots, dicots, oil crops and cruciferous seeds. The invention also provides plant seeds with a reduced level of phytic acid, suitable for the production of modified flour, and plant cells with modified inositol metabolism.

The present invention contemplates the expression of genes encoding enzymes that act on the sugar alcohol -- a precursor in the biosynthesis of phytic acid, myoinositol, causing the metabolic conversion of this precursor into dead-end or poorly converted compounds that can safely accumulate in the plant cell. In this way, a reduction in the biosynthesis of phytic acid is achieved. The methods described in the present invention may be directed to other sugar alcohols. The enzyme used to act on myoinositol may be of microbial, animal or plant origin. This enzyme can be natural or obtained by i) mutation of a known enzyme to change its specific action on a substrate, thus creating a new enzyme.

The enzyme used to act on myoinositol preferably forms compounds that are safe for the plant cell and therefore can accumulate to high levels without harmful effects on the plant cell. Within the scope of the present invention, one or more enzymes can be used, and several different properties can be used to reduce the level of myoinositol. X,

Enzymes capable of modifying myoinositol by isomerization, conjugation, phosphorylation, hydroxylation, methylation or any other similar biochemical operation or enzymes capable of removing myoinositol or other sugar alcohols are used within the scope of the present invention. at

Enzymes used in the context of the present invention can be obtained from many sources or by many methods. For example, it is possible to identify an enzyme with a known effect on chemically similar substrates. Accordingly, it is possible to identify an enzyme, isolate a gene and use it in the context of the present invention. Enzymes used in the present invention can be modified by conventional methods known in the art. "

The method is also based on the use of transformation to introduce into plant cells a gene encoding an enzyme from c. Plant cell transformation can be accomplished by many means. The methods of general application include systems based on Adgobrasiegiit, using binary and ;" of co-integrated plasmids A. (Shsptiiasiape and A. gpulodepez (e.g. (US patents 4940838 and 5464763), biolistic approach (e.g., (US patents 4945050, 5 015580 and 51496551), microinjections (e.g., US patent 4143548), direct absorption of DNA by protoplasts (e.g., |US patents Me5231019 and 54533671) or introduction by injection (e.g., (US patent 53025231). In the context of the presented invention, any method of genetic transformation of a plant cell can be used. o The method also based on the regeneration and use of plant cells or tissue with altered properties, in particular seed tissue used for feed.

Thus, plant cells with altered metabolism of sugar alcohols are obtained. From sugar alcohols

Me, numerous compounds are formed, but the most important is phytic acid or phytate. Methods of reduction

The amount of phytate in individual plant cells is actively used when using feed.

Genes encoding enzymes capable of acting on myoinosit may be natural, synthetic, or a combination thereof. One of ordinary skill in the art will readily appreciate that the coding sequence of a gene can be altered for more efficient action in plant cells. This can be done by changing the codon sequences so that they are more similar to the codon usage of the normal gene action in a plant cell. In addition, it is obvious that it is possible

Ф) to define separate areas of restrictions for convenient manipulation of the coding sequence. It is also contemplated that the addition of various sequences, such as translation enhancers, introns, etc., may be used to ensure appropriate action of the coding sequence. All these manipulations are known in this field and will be easily understood by a specialist.

It is also apparent that a tissue-selective promoter can be used to limit the action of the myoinositol-modifying enzyme to those tissues in which phytic acid is produced, such as seed tissue. Suitable seed-selective promoters include the Napin promoter from Vgavzwis pari, the phaseolin promoter from Riazeoii5, or any other seed-selective promoter. 65 In particular, within the framework of the presented invention, the use of O-methyl-transferase of myoinosit from Mezetguapipetit sguziaII pit is envisaged.

The present invention provides for the use of genetically modified seeds obtained by the action of enzymes affecting myoinositol under the control of a seed-selective promoter. Accordingly, the level of phytic acid decreases. After processing the seeds, a feed product with a reduced level of phytic acid is obtained. Thus, the production of flour with a reduced level of phytic acid is also considered as a result of using the method.

The present invention provides means of creating cruciferous seeds with a reduced level of phytic acid. In particular, the scope of the invention envisages the use of O-methyl-transferase of myoinosit, for example, from Mesetrbguapietit sguviaipit, to reduce the amount of phytic acid in cruciferous 7/o Cultures. A person skilled in the art will readily appreciate that the production of flour with a reduced level of phytic acid from cruciferous seeds can also be accomplished as a result of the present invention.

The presence of phytic acid in grain flour is known to be harmful to fish grown in aquaculture. However, some grain flours, such as rapeseed meal, have a protein composition that is useful for feeding fish. The high concentration of phytic acid in rapeseed meal limits the amount of feed that /5 Can be used in the diet of fish. Therefore, flour with a reduced level of phytic acid, obtained from cruciferous seeds, is very useful in aquaculture.

Accordingly, the presented invention finds application in the production of seeds with a changed level of phytic acid and grain feed with a reduced level of phytic acid. Modified seeds and feed are widely used, for example, for poultry, pigs, fish, ruminants and non-ruminants. It is also clear from the preceding description that any number of different enzymes can be used to modify myoinositol within the scope of the present invention. It is also apparent that various combinations of these enzymes under the control of seed-selective promoters can be used to specifically reduce phytic acid levels.

The use of the invention in the process of secondary metabolism of sugar alcohols is not limited to the listed species of plants. Virtually any type of plant that produces phytic acid can be modified by the method of the present invention. The method is useful for any agricultural crop because the enzyme i) used, O-methyl-transferase, is non-specific to all major economically important crops.

Thus, any crop used for forage or grain production that is used in whole or in part for forage can be modified in the presented manner to create plants with reduced phytate levels.

Accordingly, the method can be applied to any agricultural crop, including x, monocots (e.g., cereals, rice, wheat, barley, rye) and dicots (e.g., soybeans, cotton, alfalfa,

Vgazvisa, flax, sunflower, etc.).

With the help of the presented method, modification of both phenylpropanoid metabolism and c5 Metabolism of sugar alcohols is achieved, with specific examples of how reliably and predictably, according to the method of the presented invention, a reduction in the level of certain metabolic compounds, in particular non-food compounds, is achieved.

It is obvious to a specialist that any secondary metabolic process can be changed within the framework of the presented invention. The invention was demonstrated on two independent secondary metabolic processes and predicted and actual results were obtained. During detailed research of the invention, a reduction in the level of 7-3) of non-food factors was achieved, for example, in the cells of rapeseed, cereal, rice and cotton cells.

For example, the skilled person's attention is directed to the evaluation of the production of a separate secondary metabolite as part of the method of the present invention. Thus, one skilled in the art will readily appreciate that a basic study of the tissues in which the secondary metabolite or compound used as its precursor is synthesized, in addition to the experimentally determined time interval in which this occurs, is required. Such information will provide direction -I for selecting a promoter that will control the action of the enzyme on the secondary metabolite or its precursor. In this way, a person skilled in the art will readily realize that targeting a secondary metabolite requires an enzyme capable of acting on the precursor of this secondary metabolite in the tissue where the secondary metabolite is formed and at the time interval in which it is formed. Thus, the enzyme acting on the precursor of the above-mentioned secondary metabolite must be introduced at the time or during the synthesis of this precursor. me) Next, the specialist must perform a biochemical analysis of the tissue in which the method will be performed. This analysis

He may include determination of the level of various compounds in the tissue during the process, any compartmentalization or subcellular localization of biosynthesis, the time period at which the compound begins or ends synthesis, and any other relevant biochemical information.

When performing this analysis, the expert is able to select a promoter that provides an intensity of action suitable for the desired changes in the content of the secondary metabolite in the plant tissue. It is believed that (F) even a small change in the level of a compound used as a precursor can produce the desired effect. For example, in the partial implementation of the present invention with respect to phytic acid, one skilled in the art will readily realize that a basic study of the tissues in which phytic acid is synthesized on addition to the experimentally determined time interval in which this occurs will provide direction for selecting a promoter that will control the action of the enzyme on myoinositide. In this way, one skilled in the art will readily appreciate that targeting myoinositide requires an enzyme capable of acting on myoinositide in the tissue where it is formed phytic acid, and at the time interval in which it is produced. Thus, the enzyme acting on myoinositol must be administered at the time or during the synthesis of phytic acid. The intensity of the enzyme action can be varied to achieve a 65 defined reduction in phytic acid production This reduction can range from a few percent to almost complete removal of phytic acid to sing One skilled in the art will readily appreciate that reducing phytic acid levels, particularly in some cultures with high levels of phytic acid, may require a more intense action of the myoinositide-altering gene than in a culture where phytic acid is present in lesser amounts. Controlling the intensity of action by various means is well known. It may include the addition of DNA sequences that enhance translation, strong promoters that provide high levels of transcription, or the addition of DNA sequences such as matrix attachment or cytoskeletal attachment regions that are thought to provide reliable levels of transcription to the transgenes.

This implementation of the presented invention also provides for the creation of plant tissue, in particular plant seeds, which is used for feed. Both the direct use of seeds and the use of 7/0 Modified feed from the obtained seeds are included in the scope of the presented invention. A reduction in phytic acid levels in seeds is desirable for use in the Standards, and genetically modified seeds with altered phytic acid levels would be very useful for forage production.

In some applications, animals are fed whole grain or the grain is minimally processed but not milled. For example, cereal grain is often used for feed with minimal processing. 7/5 However, some cereals are processed and the resulting products are fed to animals, such as cereal gluten. Both types of forage can benefit from a reduction in phytic acid levels, especially when it comes to environmental degradation from excess phosphorus released by animals. For other types of grain, such as wheat, barley and oats, the presented invention can also be useful. Thus, the presented invention finds application for a wide range of economically important grain crops. The gene presented in the present invention for changing the level of myoinositol may be able to act in any cereal crop, since myoinositol is found in all cereal crops and is the only known precursor in the biosynthesis of phytic acid. Thus, the undoubted use of the methyl-transferase gene to change the level of myoinositol has been demonstrated. One skilled in the art will readily appreciate that the coding sequence of a coding region, or individual DNA sequence of a promoter, sc or untranslated region sequence, terminator, etc., can be altered to provide optimal action in a cell of a particular plant species. All this is within the capabilities of a qualified specialist. Thus, it can be confidently predicted that this invention can be carried out for any type of plant capable of genetic transformation, including those plants whose grain is used directly for the production of fodder, or those plants whose grain is first subjected to processing before using as feed. с зо Thus, as a result of using this method, fodder seeds with a reduced level of phytic acid are obtained. ise)

In addition to direct feeding of whole or partially damaged seeds, there are many ways of obtaining seed meal. Among other products, wet grinding of grain is used to obtain flour from grain gluten. In addition to processed grain products, a whole grain with a reduced level of phytic acid can be obtained according to the method of the present invention. This invention also offers the preparation of flour from grain gluten with a reduced level of phytic acid.

In addition to staple crops such as maize, oilseeds provide a large amount of vegetable meal used as feed. The production of flour during oil extraction is particularly important in the processing of oilseeds. In general, oil is extracted from oilseeds with or without a solvent. The cake or pressed solids resulting from this process are used in feed compositions, where they usually contain the majority of the phytic acid level of the seed. ;" The most widely used oilseed processing involves the extraction of the oil and the separation of the cake or final components of the seed after the oil has been extracted. The present invention proposes the use of this process

And the resulting feed components can be more valuable than the generally accepted feed, due to the reduced level of phytic acid.

Thus, the method of obtaining feed with a low level of phytic acid offers a new composition that is not contained in the feed obtained after the processing of oilseeds. The main oilseeds useful in the present invention include cruciferous oilseeds such as Vgazzisa pariz, Vgazzisa 5o "tara, Vgazzisa dipsea, Vgazzisa pidga, Zipariz aira, Statre, Egisa zaina and other cruciferous seeds

Me. oil crops that can be commercially important and used as fodder. For oil seeds

Among the non-cruciferous crops that are often used in the production of fodder are soybeans, cotton, corn, safflower, sunflower and peanuts.

The present invention contemplates the use of genetically modified oilseeds obtained as a result of the expression of enzymes capable of acting on myosinositis, where the above-mentioned enzyme is under the control of a seed-selective promoter. Thus, the amount of phytic acid is reduced. During processing

F) oil seed feed product has a reduced level of phytic acid. The following examples are provided to illustrate the method and do not limit the scope of the invention in any way.

Example 1: General methods for determining the products of the phenylpropanoid pathway: Determination of the level of sinapin, the final product of the phenylpropanoid pathway, in cruciferous seeds.

It is apparent that one skilled in the art can perform all methods of determining the products of the phenylpropanoid pathway by consulting the many analytical chemistry publications. According to the first described method, a simple test for the determination of sinapin in plant tissue, namely the tissue of seeds from cruciferous plants, was performed according to published methods. Eyiv and SK. 65 bZotegmie, 1992, Riapi SeiI 4:1413-1424). A thin-layer chromatography (TLC) protocol was standardized for the determination of sinapin, a product of the phenylpropanoid pathway. This method consisted of separating plant extracts, and a known amount of sinapin was applied to silica gel plates (for example, U/paytap bOA b5iiisab).

Sinapin can be purified using published methods (for example, O. Bzigask, 1977, 7.

RIPapgeprpusio!. 84: 139-1451). Separation was carried out due to the solvent composition of n-butanol and acetic acid

ACIDS and water in a ratio of 10:2:3, respectively, to about 7Osm from the beginning. Seed extracts were obtained by overnight infiltration of a pre-weighed sample in a tightly closed tube containing 100-B00μl of a methanol solution (9895 methanol, 2906 acetic acid), followed by the addition of the same volume of methanol and grinding. The supernatant was obtained after centrifugation at 12,000 rpm.

The supernatant was extracted with a mixture of chloroform: water (40O0µl:10µl), centrifuged as described above, 7/0 removed the lipid phase and the aqueous phase was dried in air or dried in a vacuum. Samples were dissolved in water and transferred to plates for TI C. A certain amount of the sample, determined empirically, was transferred to plates for TS before exposure to the rising solvent. In those cases where dry weight measurements were used, these measurements were made by drying and then weighing a known number of fresh samples.

The TC plate was then viewed under UV light, and sinapin was visualized as a bright spot.

A pre-purified sinapin sample was used as a single standard and also as an artificial mixture with the seed extract to ensure that the purified sinapin migrated with the sinapin in the seed extract if present together with the components of the seed extracts. When observing radioactively labeled sinapin, TS plates were scanned using an Atriz 4000 scanner (Zsapaiuiisv), which quantitatively displays radioactive radiation.

High pressure liquid chromatography (HPLC) was then used to confirm sinapin levels.

Methanolic extracts of the plant material, as described above, but without chloroform-water extraction, were subjected to NRI C using a Magiap NRI C equipped with a Misieoviya column! SI8AV. The injection volume was typically 1 µl and the mobile phase was a mixture of Solvent A (295 acetic acid in water) and Solvent B (295 acetic acid in

Acid in acetonitrile). Typical wash conditions include, in the mobile phase: Solvent A, varying from 9096 to 8095 over 17 minutes, then further falling to 1095 over 1 minute. The column was held by 1090 o

with Solvent A for four minutes, then washed and equilibrated with 9095 Solvent A. These conditions can be varied to increase the separation of eluates. For example, Solvent A can be changed from 9095 at the start of elution to 8095 for 15 min to 7095 for 15 min and then to 1095 s

Zo for 2 minutes, then held for another 2 minutes at 1095 and equilibrated at 9095. A diode detector was used to detect UV absorption. UV absorption at 30nm was used for sample analysis. at

A standard curve of purified sinapin was constructed and used to calculate sinapin in plant extracts. It is obvious to one skilled in the art that the conditions of NRI C (e.g. ratio and gradient of solvents, different types of solvents) can vary. It is desirable to determine the appropriate conditions for the equipment, and yuyu

C5 the complexity of the applied sample. Published data describe many NRI C methods for the analysis of phenolic compounds, including sinapin.

Radioactive studies were used in order to obtain a more accurate detection of the beginning of sinapin synthesis and to determine the ratio of newly synthesized sinapin in different parts of the seed. The method of radioactive determination can also be used to determine the ability of individual components of seeds (for example, cotyledons, embryos, seed coats) to synthesize sinapin from the exogenous supply of choline. Radiolabeled products can then be checked for sinapin, for example, using the TI C protocol, followed by an Atbriz4000 scan. "" The NRI C protocol was used for the quantitative analysis of the total level of sinapin in seeds and seed components. TC was used to quantify the level of sinapin at different stages of seed development, and radioactivity together with TC and subsequent radioactive spot counting was used to determine the origin and distribution of sinapin in seeds and seed tissue such as cotyledons, embryo axes, and seed coat . In the radioactive determination of the synthesis of sinapin de pomo from choline, whole pods were used, rather than isolated seeds, in order for the experimental conditions to be as close as possible to the conditions of ip mimo. Pods from 5o different stages of development were obtained from plants that were grown under control conditions -- typically 209°C daytime temperature, 1593°C night temperature with a 16/8-hour day/night cycle. Pedicel part of the pod

They were immersed in a solution containing "C-labeled choline purchased from Mem Epdiapa Misieag (mother solution activity 2.0 GBq/mmol; concentration, 7.4 MBq/ml, which is the same as 0.2 mCi/ml; choline, Z,/7 µM/ml). Pods with their stem immersed in a test tube containing 1.8 ml of an aqueous medium such as O0.5 strength medium

Mygazpide-5Kos9d, and mM of non-radioactive choline and from 1.8 to UMkl of radioactive mother solution of choline, were incubated in a plant growth chamber for 24 to 72 hours with a day/night cycle of 16 hours of light/8 hours of darkness at a temperature of 202С. The light conditions were 51 µEinsteins/m2 per second, and the seeds removed from these pods were used in the TI analysis. These samples were extracted in a methanol solution, followed by a chloroform-water (CHW) extraction as described above. 60 In those cases where older seeds were analyzed, in addition to the above-mentioned method, infiltration of seeds separated from pods was carried out. Twenty seeds were inoculated with 50O0μl of the radioactive choline medium described above under light vacuum conditions for 15 minutes at room temperature of 23,260.

The radioactive medium was then removed and replaced with 50 µl of non-radioactive medium of the above-mentioned composition, less 14C-choline and incubated at 232C for 24 hours. The seeds were then extracted as described above.

The plant material can also be extracted with a mixture of chloroform : methanol : formic acid (CME) in the ratio 5:12:3, respectively. Typically, 2 μl of CME per mg of sample was added, then the sample was ground and left at room temperature (21-232C) overnight (16 hours), after which it was centrifuged at 12,000x9 for

From minutes. The supernatant was collected and the pellet was thoroughly mixed with another volume of CME added earlier, left for 20 minutes and centrifuged as described above. Supernatants were collected and chloroform-water (CHW) extraction was performed as described above, and the aqueous fraction was assayed as described above.

Example 2: Determination of the location of synthesis of the product of the phenylpropanoid pathway in a certain tissue.

In this example, the time of synthesis of the product of the phenylpropanoid pathway during ripening was determined. This 7/0 example illustrates sinapin synthesis and accumulation within cruciferous seed tissues. In this example, the drying seeds of Vgazzievi Paris plants were used. TiC analysis of pod extracts obtained from artificially pollinated flowers was performed to determine the time of onset of sinapin accumulation. Samples were collected on 7, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 34 days after pollination (DPZ) and after seed maturation. Seed extracts were prepared according to the methods described in Example 1, and the level of sinapin was visualized by TI C analysis. Sinapin was observed from 18 DPI until seed maturity, and maturing seeds contained the highest amount of sinapin, suggesting that there is general accumulation in maturing seeds.

The pod wall fraction from which seeds were removed did not contain sinapin, and sinapin was also absent in younger seeds from samples at 7-14- and 16-DPZ. This analysis showed that the onset of sinapin accumulation was around 18 dpd, however, since this method used UV fluorescence detection, the presence of very small amounts, if this occurs in younger seeds, renders detection impossible. Fig. 3 shows an example of TI C-analysis of samples up to 28 DPH, and the next plate shows an example up to the moment of seed maturity. Since the above-mentioned results were not quantitative, NRI C-analysis of extracts from whole seeds obtained using a methanol solution was performed as described in Example 1. c

The results shown in Fig. 4 confirm the beginning of the synthesis of sinapin from 20 DPZ, with a rapid increase and duration until the moment of maturation. The amount of sinapin in the ripe seed was 0.890 on a dry weight basis o seed and since the oil level is about 4595 seed, this figure would be closer to 1.595 of the defatted seed meal.

Example 3: Determination of temporal and spatial aspects of product synthesis in the phenylpropanoid pathway -- Ha synthesis of sinapin in relation to seed maturity.

Based on what was obtained above in Example 2, it was further shown that sinapin is synthesized by the ripening seeds. It is also evident that sinapin degradation is minimal during seed ripening. To confirm this analysis, maturing seeds were incubated with "C-choline" and the precursor was used to synthesize sinapine from sinapoyl-glucose, the final step of the phenylpropanoid pathway in plants. The presence of a radioactive precursor made it possible to obtain labeled sinapin. Thus, it is possible to describe the biosynthesis of sinapin quantitatively in this way. As shown in Fig. 5, the synthesis of sinapin (inclusion of "C-choline in sinapin) was impossible to determine at 10 DPS, and becomes possible only at 14 DPS. Thus, the synthesis of sinapin (that is, the production of the final product of sinapin from the precursors of sinapoyl-glucose and choline) takes place from 14 DPH to approximately the moment of seed maturation. Thus, the beginning of the accumulation of sinapin in seeds occurs after 14 DPH, and ripening seeds continue to synthesize and accumulate sinapin until the moment of maturity. - c Although 18 DPH is the time point at which the accumulation of sinapin can be detected by non-radioactive analysis, h radioactive analysis is more sensitive and allows to detect even a small presence of biosynthesis. Thus, as shown by radioactive analysis, the synthesis of sinapin first begins at a low rate of 14

DZP Studies of infiltration show that the ability to synthesize sinapin is also characteristic of more mature seeds (Fig. b). -and In addition to determining the time frame for the accumulation of products of the phenylpropanoid pathway during ripening, one skilled in the art should further focus his efforts on determining the tissue specificity of the biosynthesis of the product of the phenylpropanoid pathway. In this part of the example, sinapin accumulation was measured in cotyledons, axils of 1 embryo and seed coat. Radioactive analysis was performed with whole pods from 18 to 42 DZ. The stalk of 50 excised pods was immersed in a solution containing "C-choline" for 24 to 72 hours, and the three seed components were excised and tested for sinapin. The results shown in Fig. 7 showed that the cotyledons contained g » the maximum amount of sinapin (relative to each seed), followed by embryo axes and seed coat.

Extracts of sinapin from cotyledons and axils of seed embryos from 25 DPH to the moment of maturity showed that the axils of the embryo contained approximately 5095 sinapin of cotyledons relative to dry weight (Fig. 8). However, since the cotyledons are relatively larger in mass compared to the embryo axes (six times greater than the mass of the axes for the embryo at

F! ripening), their sinapin level is up to 9095 of the total sinapin found in seeds (Fig. 9).

Example 4: Genetic transformation of plants containing a gene encoding an enzyme capable of acting as a precursor of the phenylpropanoid pathway.

In this example, the enzyme choline oxidase (HO), which acts on the precursor used to synthesize 60 sinapin, is expressed in a plant cell. The choline oxidase enzyme was inserted into the vector for plant transformation under the control of the seeds of a selective promoter. Choline is a precursor for the production of sinapine from sinapoyl-glucose, so a decrease in the choline pool reduces the production of sinapine.

Genetic transformation of Vgazzis paris, a cruciferous plant species, with a construct containing seed-selective choline oxidase (XO). The DNA sequence of the choline oxidase gene is shown in Figure 10, while the predicted sequence of amino acids is shown in Figure 11. In order to ensure seed-specific expression, the sequence of the parip promoter |(Koppo-Nigaze, .), M. Mygaze, N. Ispikamzha and .. was used.

Itatiga, 1994, Riapi MoiIesshiag Vioiodu, 26:1115-1124). This final pH57Z31 plasmid was constructed by serial cloning and subcloning according to standard protocols described in manuals such as (Mapiis,

T., Epivsy, E.R. and ZatrbookK, U. (1982; Moiesshiag Sioppipd:. A I aroghaiogu Mapia!: Soid Zrgipd Nagrog, Mem

Mogk))!. The vector construction comes from KO400 |Oya, K.5.5., Nattegiip!, 9. K., Rapspik, V., Reisneg I. V. and

KayPeg, MU, 1992, "Zepe 211:383-384Y"), which was modified in such a way that instead of Mo5P-MriI plant-selective marker KO400, it became a composite gene between diz and pri (biv: pr). The knock-on has already been described

Ioamtsa, K.5.5., Natitegiipa!i, 9U.K., Reispeg, .E., Stozru, MU. and b. Zeimaga), 1991, Sepe 101; 239-245). It was cloned as 2.6 kb. a fragment containing a Mov-terminator (polyA signal) at the end of the sequence at, /o with roOkKkKt4 |Oatsya ei ai., 1991) into an intermediate plasmid that has completely lost the KO400 T-DNA. Partially duplicated Samuz55 promoter together with the leader sequence of alfalfa mosaic virus with rVI525 |OSatsa, K.5.5., RE. Vekkaoci,

U.K., NatipegiipaI, p., Riiaie, O.I. Ocpviap and MUL. Stozru, 1993, Riapi Zsiepse 94; 139-149| cloned as

Nipa!p-Watni fragment to the 5-end of the sequence (ziv:pr. Nipai, Watni and adjacent Khbai sites were removed by filling the ends with a Kiep/polymerase I fragment with the formation of pHn5Z722. A functional Ias-aIrna site containing numerous cloning sites from pH719K |Meai , O.A., E. 52sgezpa-ZKogyra, and V. Ketreg, 1986, Rgoieip

Epdipeegipod 1:67-74), was obtained as 0.26 t.p.n. fragment using the polymerase chain reaction. This fragment was cleaved by MypX and EsoKiI and a site or pHn5b722 was inserted into EsoKiI to form pHn5723. The nucleotide sequence of part of the t-DNA vector was determined and unique sites in numerous cloning sites were determined by restriction analysis. pHn3725 is a derivative derived from pH5O723 and contains an open reading frame of Choline oxidase originating from pKK11 (see Kogmadomuki et al., 1991), through a series of intermediate vectors to provide convenient sites or properties such as poly A signal of cauliflower mosaic virus. The rne725 vector provides an open reading frame in rNZ723 COX along with the Samm polyA signal at the 3Z-end. The 5-part of the open reading frame containing the Samuz355 promoter was replaced by the parip promoter from the intermediate plasmid to form рН5З7З1. The intermediate plasmid contained a ruOS19 derivative containing 1.1 kbp. parip-promoter as school dv Nipap-MaripR-Vatni cassette received from u. Koppo-Mygaze | Koppo-Mygaze ei ai!., 1994), which was ligated as

Nipai!i-Watni insertion in the Nipai-Zatnii window of the KO400 plasmid |Oaya ei aiYi., 1992). The resulting plasmid i) was rn5Z9o74. The VatNi-BADH open reading frame-PolyA-Krp!I cassette was isolated as 2.1 kb. a fragment from rREKUObA (rKKbA | described in detail in K.K. Daip, 1995, Sepeiyis Epdipeehipd oi Oztoiuie Viozupipeviv ip Riapiv:

Mapirshanop oi AiIidepude Veyaipe Oepudgodepase (Sepe Ehrgezviop Torasso, R.O. Tpeziv, OpimegzPyu oi su zo Zazkaispezhmap, ZazKaiop, Sapada)) and ligated into VatNi-Krp I the window of plasmid rnz974, which led to the formation of rnZeO81. рНОЗВИ was used to create the derivative рНО7З1. Fig. 12 shows the resulting plasmid pH5Z7Z1, which contains the open reading frame of the SamM poly A signal together with the left and right parip-promoter-XO. From the components, the nucleotide sequence of the parip-promoter-HO segment of the open reading frame-SamM poly A signal was determined. Additional confirmation of the sequence is also possible, Scheo, because the precursor of pHZ7/23 included all components of rVIM19 (Wemap, M., 1984, Mysies Acids Kezeagsy 12: 1-8711-8721), including the right and left ends and the entire other region. The complete nucleotide sequence of rVIM19 has been published. Thus, the XO gene construct described above can be easily used for cloning into other vectors. Plasmid vector pH5731 in E. soybean OH5 was deposited on January 22, 1997 in

American Type Culture Collection (ATSS), 12301 Ragkiaml Yugime and KoskKmShe MO, OBA 20852, under " ATSS registration number 98300. with c Based on published material (Oye! ivIie A.)., and Stos, M.Y., 1989, Riapi Rnuzioiodu 11:617-623); . INodiyupa, A.-5., T. Kodip, E. IGagesop, and Her KavK, 1992, Riapi RPpuzioiodu, 98:509-515) and given results for the α parip promoter, it was found that the activity continues during the middle part of the term ripening of seeds

V. Paris.

The pH5Z731 vector was inserted into the Adhorasiegisht MROO strain by standard triple conjugation, after which Adhorasiegisht-mediated Vgazvis transformation was performed. The transformation was performed mainly as (described by Moiopeu ei a!., 1989, Riapi Sei! Kerogov 8:238-2421). 1 Strain Adhorasiegisht ishptiiasiepe MZ3101/rMROO (Kops2 So b BspeiY; 39).,, 1986, Moi. ep. Sepoys p 204:383-396), carrying the binary vector рН5З7З31, was used for transformation. Stationary phase bacterial culture in IV broth (Difco, USA) (100 ml) was collected by centrifugation and

Me. resuspended in 1 Oml of fresh IB-broth with 19060 OM5O (dimethyl sulfoxide) (Sigma, USA) as a cryoprotectant.

He Aliquots of 200 μl were stored at -202C until used for transformation, where the bacterial aliquot was added to 2 ml of Vgaip Neag Ipgiviop broth (Difco, USA) containing 2905 sucrose, 50 μM acetosyringone, pH 5.6 and incubated overnight at 282S. The density of bacterial cells was approximately 1 x 109 cells per ml.

Cotyledon explants were exposed to Adhobrasiegist containing a vector for plant transformation according to the method |MoiIopeu ei aiYi., 1989, Riapi Se Ker. 8:238-242). The surface from the cut side of the petiole of the explants was briefly immersed in the bacterial culture. The explants were transferred to the co-cultivation medium so that the surface from the cut side touched the medium. Ten explants were placed in each 100 x 15 mm Petri dish.

The co-cultivation dishes were hermetically closed with plastic film Zige (sp'p Zeaitm). The dishes were incubated for about three days in a growth chamber with the conditions of temperature and photoperiod as described above for the stage of seed germination. The explants were then transferred to a selective medium.

After 3-4 weeks in the selective medium, the regenerated green sprouts (original transformants) were cut out and transferred to fresh selective medium for long-term growth. When the sprouts reached a length of 1.5-2.0 cm, they were transferred to a medium for root germination. The original transgenic sprouts were screened for dis gene expression basically as (described in OePegvgop, K.A., 1987, Riapi Moi. Vioi. Ker. 5; 387-405). The presence of blue color was considered a phenomenon of transformation.

Confirmation of transformation was established using kanamycin selection, Southern blotting, PCR (polymerase chain reaction) and progeny analysis. Transgenic seeds of Vgazzis parivs, variety UUeviag, containing the vector рН5З7З31, received the number 202622 and were deposited on January 22, 1997 in the American Collection

of Typical Cultures (ATSS), 12301 RagKiamzhmp Ogime, Koskmiye MO, О5А. 20852, under the ATOS registration number

Mo978541.

Then the expression of the choline oxidase gene in the seeds of Vgazzis paris was determined. Several independent transgenic lines were regenerated. XO activity was detected using a double enzymatic reaction. XO forms betaine aldehyde from choline and this betaine aldehyde can be easily detected by NAD-dependent 7/0 oxidation of it by BADH. NAD recovery was monitored by the change in absorbance at 34 nm.

In order to standardize the analysis, different amounts of commercially available HO were used (e.g.

Sigma) and a constant amount of E. soy BADH (50 units; 1 unit-1nmol NAD reduced per minute per mg of protein) from a BADH-overexpressing bacterial strain to standardize the reaction conditions and to construct a standard curve that XO, but not BADH, is restrictive. Then the analysis was performed with plant extracts from /5 transgenic lines and controls. BADH can be saturated or purified using published methods (eg, (RaiKkepbega. R. and Zsgot, A.K., 1990, Viospetisa Viorpuzisa Asia 1034:253-2591).

Plant extracts were obtained as follows. Plant leaves weighing approximately 100 mg were frozen in liquid nitrogen, immersed in an ice buffer for extraction, two volumes per sample weight. The samples were centrifuged at 100009 and the supernatant was centrifuged again. Centrifugation was repeated,

UNTIL not a particle could be observed. Regarding the seeds, 20 seeds per sample were taken and after the first centrifugation, 20 mg of activated charcoal was added and the procedure was continued. The buffer for extraction, which had a pH of O0, which was created by adding KOH, contained 1.92 g of HERE5 per 100 ml, 0.2 ml of 0.5 MM

EOTA, 1Oml of glycerin and deionized water to bring the volume to 100ml. Before the analysis was added

OTT with 1M mother solution to a final concentration of 25MM, and a complete set of protease inhibitors was added by IVoephipdeg-Mappieit, Catalog Mo1697-498| with L1OMM of mother solution of NERE5 buffer. The buffer for enzymatic analysis (BADH buffer) contained HOMM NEREZ-KOH, pHa, 0, 1 mM EOTA and freshly added OTT to 1 mM i) final concentration. The double analysis was performed in a reaction composition containing 5 μl of BADH buffer,

BOmcl of 10MM NAD mother solution, 5Omcl of plant sample, ZOmcl or equivalent with the formation of 50 Units

BADI and deionized water to create a volume of 450 μl. Absorption was observed at 34 Ohm for 20 minutes, then 50 μl of choline chloride was added and the spectrophotometric study lasted from 10 to 20 minutes.

For the purpose of constructing a standard curve, a plant sample was not used and instead, varying amounts of purified HO were added. Units were counted using a spectrophotometer with software control of the Vesktap BYb65 type. One of ordinary skill in the art can modify the protocols for existing equipment using biochemical data existing in the literature regarding the extinction coefficient of NAD. Recovery of NAD in itself is a standard assay performed for dehydrogenases. Cha

Example 5: Study of transgenic seeds for a reduced level of phenolic compounds

In this example, the seeds were tested for sinapin levels. Seeds from transgenic plants obtained in

Example 4, were grown to obtain self-pollinated progeny, and the lines of these progeny were examined for segregation or absence of a transgene encoding Dr-glucuronidase activity (305). This gene | see Oaya, « 0 B.5.5., Nattepipa!, U.K., Reisneg, G.E., Stozru, M.H., and b. zeimaga), 1991, Sepe 101:239-246) is present in seh) with the limits of the right and left ends of the t-DNA vector рН5З7З31 and, thus, is a convenient marker for genetic and analytical segregation. The absence of genetic segregation testified to the homozygous nature of the plant from which "" the offspring originate. Those lines that showed segregation were hemizygotes, and segregants that had no activity were kept as controls that did not contain the XO gene. Thus, homozygous lines containing

HO, and lines that did not contain the transgene. These lines were examined for the level of sinapin using the protocol of NRI S. -i The results presented in Fig.13 show that the transgenic segregants have a significantly reduced amount of sinapin compared to their non-transgenic counterparts. Non-transgenic segregants are better controlled because they are derived from a single parent transgenic plant. These results show the use of the described method. It will be apparent to one of ordinary skill in the art that varying amounts of reduction may be accompanied by variation in the amount, timing, and location of gene expression, as well as the search for beneficial variants among individual transgenic lines that exhibit desired results for a variety of reasons, including location and amount. copies

Example 6. Generation of a stress-protected product by altering a precursor within the phenylpropanoid pathway.

In this example, betaine was synthesized as a stress defense through the binding activity of XO and BADH, both under the control of a seed-selective promoter. As in the first part of this example, iF) transgenic Vgazzis paris were created, carrying the BADH gene under the control of the seeds of a selective promoter. Analysis of these plants for BADH expression was carried out.

Betaine aldehyde dehydrogenase gene expression in Vgazzis paris seeds. The BADH gene of E. soybean (reiB) was isolated and prepared for seed-specific expression by combining with the parip promoter of B. paris |Voua ei ai.,

Sep 103:45-52 (1990). Plasmid KO400 |OSaitsa, kK.5.5., Nattegiimva!, 9U.K.. RapspikK, V., Reisneg, I.E., and

Keyeg, MU., 1992, Sepe 211:383-384| was used as a vector from which the final derivative pRN5ZO74 was obtained, containing within the right and left ends of the T-DNA an open reading frame of bBeiB - from its ATO site under the control of the expression of the parip promoter and the polyA signal of the cauliflower mosaic virus. The Marip-be(B-PolyA cassette (3.3 b.p.n.r.) contained in the specified sequence: the Nipaiia site, Marip-promoter, WatNi site, re(V OKR, Esoki site-PolyA signal of cauliflower mosaic virus DNA, Krpi site and EsokKiI site Marip-promoter was from

Koppo-Migaze ei ai. (Reapi MoiIesshag Vioiodu, 26:115-1124, 1994) and the PolyA signal was originally from the plasmid ru!/T1117 IScegipeai, R., MUsoiviop, 5., VgooKk5, |. and Mijpeaich, 1988, Mysies Acids Kezeagsp 16:11380).

The final plasmid pH5Zo81 15 t.p.n. shown in Fig.14. This plasmid was introduced into Adgorasiegiit (Shsptegasiepe SMZ3101

IRMROOY IKopsg, S, and ZsNpeiY, 9U., 1986, MoiIesciag and Sepega! Sepeiyisv 204: 383-396| and the resulting strain was used for genetic transformation of Vgazvis pari.

Several transgenic lines were obtained and seeds at different stages of development were examined for BADH activity.

BADH activity was maximal at about 35 DPI, and ripe seeds left the final activity.

Specific activity as a function of maturation stage showed that the onset of BADH activity coincides with the synthesis of 7/o binapine.

Lines expressing the BADH gene were crossed with lines carrying the XO gene, as described in Example 4. Plants containing the XO gene and plants containing both the BADH and XO genes were examined for the level of sinapin and the total level of phenolic compounds. These results are shown in Fig. 15 (level of sinapin) and Fig. 1b6 (total level of phenolic compounds). The studied seeds were collected from plants and cultivated in the field in the summer of 1999. As shown in Fig

Fig. 15, the combined activity of XO and BADH led to a further decrease in the accumulation of sinapin during the activity of only one XO. As shown in Fig. 1b6, there is a decrease in the total content of phenolic compounds in transgenic plants relative to the control.

Example 7: Nucleotide sequence of the synthetic gene of ferulic acid decarboxylase.

In this example, the published sequence of ferulic acid decarboxylase from Vasiy5 ritishz (7ado ei 2o a. Arrie4y and Eepmigoptepia! Mishoriojudu 61:4484-4486, 1995| was used to create a gene optimized for expression in plant cells. The open reading frame of ferulic acid decarboxylase was synthesized by by combining synthetic oligonucleotides based on the published sequence Oligonucleotides were synthesized based mainly on the predominant codons of overexpressed genes

The synthetic oligonucleotides were approximately 60 nucleotides in length. The structure of oligonucleotide duplexes included at the 5-end VatNi-sticky end (5 (zZATS-) and EdoK! sticky end (3-TTAA-5) at the 3'-end. Separate duplexes were assembled and a full-length open reading frame was formed with 5' Watni and Z EsokKiI sites.

Binding reactions were performed according to standard protocols. The ligation products, in turn, were ligated into a vector for cloning. When the aforementioned synthetic gene was added to VatNi-EsokKiI cleaved rVishpevzstgiri 5K- (Zia(adepe), clones with a 0.5 kb insert were determined by screening DNA plasmids from E. soybean clones. Potential candidates were screened and determined the nucleotide sequence of two of the clones It was found that each of the clones had a point mutation, and these two point mutations were different in location in the two selected clones.

The distance between these mutations made it possible to reconstruct the intact gene by connecting the two non-mutated parts of the gene to the ZHI clones. This clone was named pOoz97p1. The nucleotide sequence of this synthetic gene is shown on

Fig. 17. The predicted sequence of amino acids is shown in Fig.18.

The functionality of the synthetic gene was tested using a simple test. Ferulic acid decarboxylase converts ferulic acid to 4-vinylviacol (4-MO). 4-MO has a distinct clove smell and is considered to be the single most important compound that imparts the natural aroma of cloves. The Ezspegisnia soybean strain with ROSZU9T1 when grown in the presence of TMM ferulic acid in the growth medium produced a distinct clove odor, whereas the culture without ferulic acid or the culture of the vector-only strain had no odor. NRIS analysis of ferulic acid uptake cultures confirmed the disappearance of ferulic acid. Based on ;" of the above-mentioned results, it can be concluded that the functional gene is cloned in roz97r1.

Example 8: Genetic transformation of a plant carrying a gene encoding ferulic acid decarboxylase

Under the control of the constitutive promoter -I. In this example, the enzyme ferulic acid decarboxylase cloned in ro597r1 was inserted into the vector for plant transformation KO400O | .E., apa Keieg, MU., about 1992, Sepe 211:383-384)|, which was modified in such a way that instead of MozR-Mri, the plant-selective marker KO400 contained a fused gene between div and pri (biv:pr ). biv-pri was previously described |Oaia, MK.5.5.,

Nattegiipai, 9U.K., Reiispeg, G.E., Stozru, MU.Yi. and b. BZeimaga), 1991, Sepe 211: 239-246). Decarboxylase gene

Me, ferulic acid was placed under the control of the 355 promoter and the plasmid was used for

From the transformation of tobacco plants according to standard protocols.

The restriction map of the vector is shown in Fig.19. Vector roz97p3, as shown in Fig. 19, is the same as rNOZ'31, except for the following. On the same vector basis рНб7З1 NipapP-parip R-Watni cassette ov was replaced by the cassette: 355 promoter of cauliflower mosaic virus-leader sequence of alfalfa mosaic virus is described in K.5.5. Yuaya, ER. VekKKaotsi, U.K. Nattegiip!, p. Riyaie, O.I. Yuipeiap apa MUL. Stovru Itrgomei

F) subp-Ieme! sopviyshime Togedp depe ehrgezviop ip riapiv izvipd ap AMM KMA4 ipigapziaie (y Iesadeg zedpepse, ka Riapi Zsiepse 94: 139-149, 1993). This part of the promoter received the abbreviated name 355 in Fig.19. After 355, there is an open reading frame of ferulic acid decarboxylase, connected by VatNi at the C-terminus and Esok at the C'-terminus instead of the open reading frame of ХО рнз7з31.

Transgenic plants expressing the ferulic acid decarboxylase gene were discovered. The plants were examined for the level of phenolic compounds and the release of vinyl guaiacol.

Using Western blot analysis using polyclonal anti-ferulic acid decarboxylase antibodies, independent transgenic tobacco plants carrying the ferulic acid decarboxylase 65 gene were found to contain an immunoreactive polypeptide of the expected size. Non-transformed plants had no immunoreactive peptide. This confirms the fact of transgenic expression of ferulic acid decarboxylase protein in these transgenic plants.

Example 9: Genetic transformation of a plant containing a gene encoding ferulic acid decarboxylase under the control of a tissue-selective promoter.

In this example, the enzyme ferulic acid decarboxylase, cloned in ro597p1, was inserted into the vector

KO400 for plant transformation. The ferulic acid decarboxylase gene was placed under the control of a seed-specific parip promoter from B. paris and then the plasmid was used to transform tobacco plants according to standard protocols. The restrictive small vector is shown in Fig. 20. Vector roz97p2, as shown in Fig. 20, is the same as p597p3, except that on the same vector 7/0. The Nipap-355-Watni cassette was replaced by the NipaI-parip promoter-Watni cassette, shown in Fig. 12 for pHn5e731.

Transgenic plants expressing the ferulic acid decarboxylase gene were discovered. The plants were examined for the level of phenolic compounds and the release of vinyl guaiacol.

Example 10. Determination of tissue specificity and accumulation of phytic acid in seeds during ripening of the seeds of the cruciferous plant, Vgazvisa pariv.

Biosynthesis of phytic acid.

In this example, data on the biosynthetic pathway of phytic acid were determined in Vgazvis seeds.

Since phytic acid is a hexaphosphate derivative of myoinositol (phytic acid is denoted as IR b), the ratio of individual phosphorylated derivatives of myoinositol (for example, IR 4, IR», IR3, IR, and IRb) to the total derivatives of phosphorylated inositol was determined. Pure phytic acid, partially destroyed by 2o autoclaving to obtain various phosphorylated derivatives, was used as a standard for analysis

NRGIS conducted according to Megop and Voppegi (1992. Tpe EMVO doigpa! 11:2077-2085) and Megpop OM, Taggsgupeki MS and Udepzep KO and Voppegi Nu), (1993. Tne Riapi doigpai 4: 199-205.

Different forms of phosphorylated inositol (eg IR, IR, IR3, IR; and IRb5) and phytic acid were easily distinguished by this assay. Only one peak was found and corresponded to IR b (ie, phytic acid) when samples of inositol phosphate extracted from ripening Vgazvis seeds were analyzed. This result showed that IRe is the main form of inositol phosphate in ripening seeds, and that other intermediate i) phosphorylated forms of inositol could not be detected using HRI C by the described methods. Thus, it is believed that the biosynthesis of phytic acid from myoinositol occurs rapidly and mainly quantitatively.

Biosynthesis of phytic acid during ripening. с зо In this part of the example, the accumulation of phytic acid during seed ripening was determined. The methods of determining the level of phytic acid in seeds were as follows:

The seeds were ground in a mortar in the presence of liquid nitrogen and the powder was transferred to a 15-ml sterile test tube containing 5 ml of 0.5 M NOI. After removal of lipids by hexane extraction, the remaining phase (aqueous phase with tissue remnants) was sonicated for 90 seconds at Z ultrasonic o liquid processor |Mode! XI/2020, Neaii Zuvietv, Ips., Farmingdale, NY, USA. After centrifugation, the liquid was transferred to a fresh test tube for phytic acid analysis using NRI C. This method was applied to seeds at different stages of maturity.

Accumulation of phytic acid during seed ripening is shown in Fig. 21. Although phytic acid can initially be observed in seeds at very early stages (ie, 12 days after pollination), its level does not increase significantly until 22 days after pollination. During the 10-day period after its first appearance with c, phytic acid reaches a maximum level of approximately 24 Ωkg/seed. The level of phytic acid relative to dry weight of ripe seeds was approximately 3.295. These results indicate that a decrease in the amount of phytin; acid due to the interaction with the biosynthesis of phytic acid requires a promoter capable of expressing the gene from approximately 12 dp to seed maturity.

Further analysis showed that phytic acid mainly accumulates in the embryonic tissue -I more than in the seed coat (Fig. 22). Cotyledons contain almost 9095 phytic acid seeds and 1095 are present in the germ axis. These studies suggest that various embryonic tissues are the most preferred target tissues for reducing phytic acid levels by genetic modification of phytic acid biosynthesis.

Example 11. Determination of the metabolism of myoinosit in the tissues of ripening seeds.

Me, This example illustrates a portion of the myoinositol pool used for phytic acid biosynthesis. Although

Hemyoinositol is a precursor of phytic acid synthesis in plants, it is also used in other anabolic pathways to produce phosphatidylinositol and cell wall components. This example illustrates a portion of the total myoinositol pool used for phytic acid biosynthesis in maturing seeds. Marking of Vgazvis ip mimo seeds, using ZN-myoinosite, was used to observe the distribution of inositol in different fractions extracted with different solvents. The method used

IF) for pulse marking of ripening ip mimo seeds with ZH-myoinositol, described as follows. Pods at various stages of development were removed, and the cut end was immediately transferred to 10 ml of sterile culture medium containing 5 μCi of ZN-myoinositol in a 50-ml tube and cultured under standard 60 growth conditions (202 for 16 hours in the light , 1593 for 8 hours without light) for two days. The seeds were collected for the extraction of lipids, phytic acids, trifluoroacetic acid (TPA)-soluble cell wall components, and cell wall fragments. Radioactivity was determined in each fraction using a liquid scintillation counter.

Four types of extractions were performed from seeds with pulse labeling to separate water-soluble cell components (for phytic acid analysis), hexane-soluble (lipid analysis), trifluoroacetic acid (TERA)-soluble cell wall components and cell debris. Figure 23 shows the average radioactivity in each fraction at different stages of seed development. The data indicate that more than 2090 of the total level of the label in the seed is in the lipid fraction at 25-30 days after pollination (DPZ).

The radioactivity in the cell wall fractions (TERA-soluble cell wall and cell debris) is approximately 595 of the total level of label incorporated during seed development. The radioactivity in the water-soluble fraction was further analyzed using NRI C to determine the portion of phytic acid. It was found that about 1095 of the label in the water-soluble extract was in the phytic acid pic and about 3090 of the label was in the pic of the injection sample, which is free myoinositol. Other labeled material present in the water-soluble extract was isolated in the pre- and post-peaks of phytic acid representing unidentified 7/0 compounds.

The percentage of metabolized label found in the phytic acid, lipid, TPA-soluble cell wall, and cell debris fractions is shown in Figure 3. Approximately 3095 labels of ZN-myoinositol-derived metabolites are found in phytic acid at 20-30 DPI. At the same time, approximately 6095 labels were present in lipids (inositol-containing phospholipids) and less than 1095 in the cell wall fraction.

Thus, the fraction of the total myoinositol pool used for phytic acid biosynthesis is approximately 3095 during the period of seed development when phytic acid biosynthesis is maximal.

Example 12. Cloning of a gene encoding an enzyme capable of acting on myoinosit.

In this example, a gene capable of acting on myoinositis was isolated. The gene was an inositol O-methyl-transferase gene from common plants of the Ai2goaceae family. Reverse transcriptase cloning was used to isolate the gene as described below. Standard DNA procedures were performed according to

Moiiesshiag Siopipo, A I arogaiyugu Mapiai, Soia Zrgipd Nagbrog I arogayugu, Soiy Zrgipd Nagbrog, MM.

Total RNA was extracted from treated 500 MM mass of Aigoaceae leaf tissues and purified by poly(AHARNA. This

mRNA was used for reverse transcription with Ziregzstgiri II Kemegze Tgapsgiriaze (Rgoteda,

Madizop, UMI.) under the following conditions: three μg of mRNA dissolved in 20 μl of water were used as a matrix. Ha RNA was denatured by heating at 652 for 5 minutes, then cooled on ice. Zmkl oligo-d was added to the composition! (50 Ωkg/ml), 1 μl of 5X reverse transcriptase buffer, 4 μl of 01 M OT, 2 μl of 10 mM o dHtTPates. The mixture was heated to 422C, then Zmkl (60 units) of reverse transcriptase Ziregigapzsgiri was added

II. The reaction was carried out for one hour at 4220. After this period of time, 1 µl of RNase H (1.50/µl) was added and the reaction was carried out for 30 minutes at 3720. The resulting cDNA was used as a template for PCR using Mapi polymerase as follows conditions: the ROK reaction was performed in a volume of 100 Ωcl with cycles consisting of 949 for a minute, 5529 for a minute, 729С for a minute with 2-second extensions during each cycle for a total of 30 cycles. The primers used in this reaction were based on the published IMT DNA sequence (SepVapk accession number M87340). IN

The forward primer used was the sequence of I.O. Mo. 6:

UTTTTTObATSSATOASTASTTASASAATOOSSAASTASA Z - which contains a Watney site at the b5'-end (underlined) The reverse primer used was the sequence of I.O. Mo. 7:

UTTTTTTTTTOSOOSSOSATAADASOSASAADATSATASASTO « du which contains the site My! at the 5'-end (underlined). with

The amplified DNA fragment was cleaved by Watn I and Moi I and then subcloned into RZROKT 1 (VK, VeiNegzaa, c MO). The cleaved Vat NI, Moi I fragment of ROK was cloned into the corresponding sites of the pZROKT vector with the formation of :z» rarogiMtT. Figure 24 (Sequence I.O. Mo.5) shows the sequence of the amplified DNA fragment, which is identical to the published IMT DNA sequence with the exception of two bases in the 3'-untranslated region.

The predicted protein sequence is identical to the published data from SepeVapk. - 15 Example 13: Creation of vectors for the transformation of plants containing a gene that encodes an enzyme capable of acting on myoinositol: use of the myoinositol O-methyltransferase Aigoaceae gene. 1 This example illustrates the creation of a vector for plant transformation containing a gene capable of acting on sl myoinosit under the control of a promoter active in plant cells. The given vector for plant transformation contains the missinosit O-methyltransferase gene under the control of a promoter active in seed cells, i.e. 355 (o) 50 promoter. The vector was created as follows: reropiMT was cleaved by Vaip NI and Eso KI to release the gene "from IMT. The IMT gene fragment was cloned into the corresponding sites of rVishezsgiri 5ZK(-), IZigayedepe, ia oia, SA. and the resulting plasmid was named rVishez!MT pViieMT was cleaved with ZreI and cloned into the vector rVI 221 (SiopTesi), previously cut by Xra I. The plasmid containing the BMI gene in the correct orientation relative to the 355 promoter in rVI 221 was selected, and Nipa PSh and Eso KI were cleaved. Fragment 355-IMT - Moz terminator was transferred into pKO 400, which led to the creation of a vector for plant transformation pZ55IMT.

GF) The resulting construct, pZ355IMT, contains the cassette: 355 promoter-IMT-505-Moz terminator in pKOraob0O and 7 is shown in Fig.25.

Example 14: Creation of vectors for the transformation of plants containing a gene encoding an enzyme capable of acting on myoinositol under the control of a seed selective promoter. 60 In this example, a gene encoding an enzyme capable of acting on myoinosit under the control of a seed-selective promoter was created in a vector for plant transformation. The gene used was the myoinosit O-methyltransferase gene and the promoter was a seed-selective parip promoter. The vector was named

RMIMT

The rMIMT vector was constructed as follows: The Zre I-cleaved fragment of IMGDNA (Example 4) was ligated into the pONI and Iparip promoter, as described by Koppo-Migaze et al., 1994, Riapi MoiIesshiag Vioidou, 26:1115-1124)) at the Khbeya site , which led to the formation of the parip-promoter-IMT-Moz terminator cassette. This expression cassette was then transferred to RKO400. The resulting vector is shown in Fig.26.

Example 15: Transformation of Vgazvis Paris (U/eviag) using RZ5BIMT.

The pZ355IMT vector was inserted into the Adgobrasiegisht MROO strain using a standard triple conjugation, followed by Adgorasiegisht-mediated Vgazzis transformation. Transformation was performed as described in Example 4. Plants transformed with pZ3551IMT were obtained and tested for phytate levels.

Example 16: Molecular analysis of transgenic plants

Transgenic ER plants containing the Samm 355-IMT expression cassette were analyzed by RSC, 7/0 Southern and Northern blots, and BMI enzyme analysis. Preliminary analysis of RSC showed that all transgenic plants contained the BMI gene (Fig. 27). Southern hybridization showed that the BMI gene was integrated into the Vgazvis genome with different copy numbers. Total RNA was extracted from maturing seeds for northern hybridization, as shown in Fig.28.

Example 17: Analysis of phytic acid in transgenic plants.

The level of phytic acid was determined in ripe seeds collected from ER of transgenic plants. Seed extraction was performed as described in Example 10. The collected data show that an average reduction of more than 1595 phytic acid is found in transgenic plants. Figures 29 and 30 show these data. The collected data show that an average reduction of more than 1595 phytic acid is found in transgenic plants containing the reIMT vector. En-seeds are compositions of transgenic and non-transgenic seeds due to 2o segregation, so the true reduction in phytic acid levels is significantly higher per transformed seed. PE" and Ez seeds are homozygous lines containing the rzIMT vector. It is obvious that in field conditions, homozygous lines carrying rzIMT vector showed an average of 3096 decrease in the level of phytic acid in seeds. In addition to determining the level of phytic acid, Southern blot analysis was performed to determine the number of copies of the inserted genes. Even in plants with one gene insertion, a significant reduction (3470) of phytic acid is observed (for example, plant MeTRI1). Thus, the expression of the enzyme is able to modify the myoinosit in the tissues that are responsible for the biosynthesis of phytic acid, which leads to (8) a decrease in the amount of phytic acid in the seed. It is also understood that the low phytic acid trait can be sexually transmitted since it is inherited, so the method of use can also include passing on the trait by conventional breeding methods once the low phytic acid trait is established.

Example 18: Transformation of rMIMT Vgazvisa passive. ise)

The pZ5ZMIMT vector was inserted into Adhorasiegist strain MROO using a standard triple conjugation, followed by Adhorasiegist-mediated Vgazzis transformation. The transformation was performed as described in Example 4. o

Plants transformed with pZ5MIMT were obtained and tested for reduced phytic acid levels as described in Example 17. Figure 31 shows a table of data collected from E; and P» of the transgenic seed bearing

RMIMT vector grown in field conditions. In E plants, the rMIMT insertion segregated, so the seeds tested were a mixture of transgenic and non-transgenic segregants. In the E5 generation, most of the lines were homozygous or nearly homozygous. In analysis 2, almost 4095 decrease in the level of phytin "

Acids were observed in seeds from plants grown in the field. Thus, the expression of an enzyme capable of reducing the availability of mysinosite for the biosynthesis of phytic acid in a seed-selective manner leads to a significant decrease in the amount of phytic acid. ;" Example 19: The formation and accumulation of UDP-galactose is not allowed by using an enzyme that is foreign to plant cells.

The enzyme UDP-galactose 4-epimerase (daiE) is involved in the main stages of galactose metabolism in living -I systems. It catalyzes the conversion of UDF-galactose into UDF-glucose. The enzyme gene is available from humans, yeast and bacteria. o The purpose of this example is to overexpress this enzyme in certain tissues of the target plant in order to increase the pool of UDP-glucose at the expense of UDP-galactose. The predicted result is reduced biosynthesis of galactinol, which is a precursor to non-food sucrose glycosides (KEO-glycosides (raffinose family of oligosaccharides),

Me, for example, raffinose, stachyose, etc.). In order to achieve this goal, it is necessary to reduce the rate of accumulation

He of undesirable KEO-glycosides, which would lead to increased availability of UDP-glucose and sucrose at the same time. The latter should participate and enhance other metabolic pathways where sucrose is needed, either for the formation of other metabolites that are necessary for the plant, or as a carbon source for increased plant productivity (eg proteins, lipids, total yield, etc.).

This example illustrates the use of a bacterial enzyme in higher plants to induce metabolic

F) transformation of one of the necessary substrates into another, while the new substrate formed will be easily used by the plant in many important metabolic transformations.

Example 20: Changing the amount of sugar derivatives using the enzyme phosphoglucomutase (FGM). The enzyme phosphoglucomutase (FGM) catalyzes the conversion of glucose ((cI)-1- and (Ic-b6)-phosphate in the synthesis and absorption of sucrose. The enzyme plays a major role in the synthesis and utilization of sucrose, starch, and glycogen, and is present in all organisms. The gene of this enzyme is available from many eukaryotic as well as bacterial sources (e.g. Adhorasiegiit).

Glucose-1-F and Glucose-6-F are essential substrates in several major carbohydrate metabolic pathways in 65 all living organisms. In particular, Glucose-1-F is the main substrate for obtaining UDPf-glucose, which is the main substrate of sucrose biosynthesis. Glucose-6-F, on the other hand, is the main starting material for several interconversions of sugars, one of which is the synthesis of myoinosit-1-P. The latter is the main substrate and cofactor in the synthesis of phytic acid and KEO-sugars, respectively.

The purpose of this example is that with the help of overexpression of the bacterial gene ROM in the target plant, the relative ratio of Glucose-1-F to Glucose-6-F can be changed in favor of one or the other of the two phosphorylated forms of glucose (tissue-dependent). By properly directing this activity, for example, in maturing seeds (accumulating tissues), it is possible to predict an increase in the number of

Glucose-1-F, which will be needed to obtain UDP- or ADPf-glucose and, later, sucrose and other reserve substances, such as proteins, lipids or starch. On the other hand, reducing the amount of Glucose-6-F will lead to 7/0 Reduction in the amount of non-nutritional factors mentioned above.

Example 21: Creation and regeneration of transgenic corn cells.

Type I callus cultures! were initiated from immature zygotic embryos of the Ni-I genotype KAgtvigopd ei ai., (1991) Maige Sepei. Soor. Memuviei, 65; 92-93) Immature embryos were isolated approximately 14 days after pollination from greenhouse-grown cobs from crosses between Ni-II parent plant A and NiII-II /5 parent plant B or Go embryos from self-pollination or pollination among sibs of Ni-II plants. Immature embryos (1.5--3.5 mm) were cultured in medium for initiation containing Mb salts and vitamins (Spi ei ai., (1978) Tpe

Mb tediit apa iv arriisayop (o apoiyeg syyige oi segeaI! sgorv. Rgos. Zutr. Riapi Tizvye Siyshge, Recipd

Egeez, 43-56), 1.0mg/L 2,4-O0, 25mMM I-proline, 10Omg/l casein hydrolyzate, TOmg/l Ao9MOz, 2.5g/l SE KITE

Izspmeiigegpa!i, Zotii Riaipiei, ML, and 20Og/l sucrose with pH5.8. After 4-6 weeks, the callus was subcultured on growth medium (initiation medium in which AOMO c is absent and I-proline is reduced to bmM). Selection for type II callus was performed after 12-16 weeks.

For bombardment or introduction of foreign DNA into plant cells (transformation by microparticle bombardment), 14 µg of plasmid DNA (for example, a vector containing 355-RAT/maize globulin promoter//MT) was precipitated on 60 mg spherical gold particles washed with alcohol (1 ,5--3,O0μm in diameter, Aiagisn sch

Spetis! So., Ips., Mim"acKee, MI), by adding 74 μl of 2.5 M SasSi" H2O and 30 μl of 0.1 M spermidine (free base) to 30 μl of plasmid DNA and H2O. The solution was immediately shaken, and the gold particles coated DNA, (8) were precipitated. The resulting clear supernatant was removed, and the gold particles were resuspended in 1 ml of absolute ethanol. This suspension was diluted with absolute ethanol to obtain 15 mg of DNA-coated gold/ml.

It is better if the plasmid DNA used contains a selective marker, such as the Bag or rai gene, which confers resistance to phosphinothricin or the herbicide glufosinate ammonium, and a genetic construct capable of altering secondary metabolism, such as the coding region of the BMI gene described in Fig. 24 or the coding region of the ferulic acid decarboxylase gene described in Figure 17 under the control of a suitable seed-selective promoter, such as the maize globulin 1 promoter or the maize zein promoter. Alternatively, a constitutive promoter can be used, such as the maize ubiquitin promoter or the rice actin promoter o

Sv FOR the expression of BMI or ferulic acid decarboxylase. uh-

Approximately bOOmg of embryogenic callus tissue was distributed along the surface of type II callus growth medium without casein hydrolyzate and I-proline, but with 0.2M sorbitol and 0.2M mannitol as osmotic agents. The callus was kept for 4 hours as pre-treated and then transferred to culture vessels containing a medium for bombardment (osmotic medium thickened with 20 g/l TS "agar (RpuyutTesppoiodu Garogaiyuogies, GIS, Zpamupee Mivviop, KZ) instead of 7 g/l). ZEKITE. Helium gas (as the active force) was used to accelerate the suspended DNA-coated gold particles toward and into the target tissues. The device used is described in US Pat. No. 5,141,131, which is incorporated herein by reference. The tissues were covered with a stainless steel plate. steel (104 µm holes) and transferred to a partial vacuum of 62.5 mm Hg in the chamber of the device. The DNA-coated gold particles were diluted 1:11 with absolute ethanol before bombardment and bombarded the callus four times using a helium pressure of 10.342 MPa, -and bombarding each time 2 µl DNA/gold suspension Immediately after bombardment, tissues were transferred to osmotic medium for recovery for 16-24 hours. This tissue was cut into small pieces and transferred to a selective medium (medium for growth without casein hydrolyzate and cY-proline, but with ZOmg/l VABTAF (AagEmo, Vegiip, Septapu). Every four weeks for 3 months, pieces of 5-year tissues were nonselectively transferred to a fresh selective medium. After / weeks and up to 22 weeks sectors

Me, the callus that showed proliferation was removed and separated. VAVTAF - resistant tissues formed,

He was subcultured every two weeks on fresh selective medium. After appropriate analysis, positive transgenic lines were identified and transferred to the medium for regeneration.

Regeneration was initiated by transferring callus tissues to media for inducing with two Cytokines, which consists of Mygazpide and ZKosd salts - hereinafter M5-salts, and Mygazpide apa ZKoso vitamins, (1962)

Ryuzio!i Riapi 15:473-497| 300 mg/l of sucrose, 100 mg/l of myoinositol, 300 mg/l of mannitol, 5 mg/l of b-benzylaminopurine, --

F) then VAR, 0.025mg/l 2,4-O0, ZOmg/l BA5TA and 2.5g/l SEI KITE at pH5.7. Cultures were placed under low ka light (125Kd) for one week, followed by one week under bright light (325Kd). After a two-week induction period, tissues were transferred indiscriminately to hormone-free regeneration medium identical to the induction medium except that it lacked 2,4-O0 and VAR and maintained under brilliant light. Small (1.5-33 cm) rings were removed and transferred to 150x525 mm vessels for cultivation, containing ZN-medium (ZN-salts and vitamins (ZspepkK apa Niiidegbapai, (1972) Sap. 9. Voi. 50:199-204)) )

Larger rings were transferred to 12 cm pots containing approximately 0.25 kg of METKO-MIH 360 (Te Zsoynv So. 65 MaguzmuShe, ON) in the greenhouse as soon as they began to grow and developed a sufficient root system. The rings were grown under a 16-hour photoperiod created by a combination of high-pressure sodium and metal halide lamps, and watered as needed with a combination of three independent Reyege Echse fertilizer formulations! (Sgase-Zieta Nopisiyiga! Rgodisiv Sotrapu, Miiryaz, SA). When 6-8 leaves appeared, the plants were transplanted into 18.9-liter pots containing approximately 4 kg of METKO-MIX 360 and grown to maturity

Voslin of transgenic corn. The seeds from these plants contained genes inserted into immature embryos, and when grown, the plants expressed the proteins encoded by the inserted DNA.

Example 22: Creation of transgenic rice.

To initiate embryogenic callus, ripe seeds of the Japanese variety Tairei 309 were cleaned and subjected to surface sterilization with 7095 ethanol for 2-5 minutes, after which they were soaked for 30-45 minutes in 5090 76 commercial bleach (2.695 sodium hypochlorite) with a few drops of IGidtsipoh soap. Seeds were then washed three times with sterile distilled water and transferred to filter paper before being transferred to callus induction medium (ie, MV). The MV environment consists of Mb macroelements (Spy, 1978,

Mb medium and its application to the culture of anthers of grain crops. (Rhos. Zugpr. Riapi Tizzie Sypyge,

RecKipd Rgezz, p. 43-56Y), B5 microelements and vitamins | (Satrogud ei aiY. 1968, Miygiepi gedzigetepiv oi 7/5 Vyzrepviop syMygezi oi voureap gooi seiv. Ehr. Sei Kev. 50: 151-158), З0Omg/l casein hydrolyzate, 500 mg/l I-proline, 500 mg/l I-glutamine, 30 g/l sucrose, 2 mg/l 2,4-dichloro-phenoxyacetic acid (2,4-Y3) and 2.5 g/l more (Zspueigegnaii, MO) with pH-5.8. Ripe seeds cultivated on callus medium for induction were incubated in the dark at 232C. After 3 weeks of cultivation, the main callus formed from the shield area of a ripe embryo was transferred to fresh MV medium for further cultivation.

We used the transformation of Violiviis plant tissues for the introduction of foreign DNA. Approximately 14 µg of plasmid DNA was precipitated onto bOmg 1.0 µm (Vis-Kai) gold particles as described in Example 21.

A plasmid was used that contained the maize ubiquitin promoter driving pre (hygromycin phosphotransferase) and the maize ubiquitin 1, maize globulin 1, or zein promoter driving the BMI gene. Approximately 14µg of plasmid DNA was deposited onto 1.µm bomg (Vio-Cad) gold particles as described herein.

For helium bombardment, actively growing embryogenic callus cultures 2-4 mm in size were subjected to extensive osmotic treatment. This treatment included placing the callus on MV medium with 0.2M mannitol and 02M sorbitol. 88| for 4 hours before helium bombardment. After gas osmotic treatment, the callus cultures were transferred to the bombardment medium (MV4-290 agar) and covered with a stainless steel plate (230μm). The callus cultures were bombarded under a pressure of 13.789 MPa twice on the target. After bombarded callus were transferred back to high osmotic medium overnight before transfer to selective medium containing MV medium with 30 mg/L hygromycin.

After 2 weeks, the cultures were transferred to a fresh selective medium with a higher concentration of the selective agent

Зз5 of the agent, i.e. МВжб5Омг/l hygromycin МКС ек а. 1993) Ap itrgomei gise (gapviogta (yop zvuviet ivipd (She Kk

Brioiiviis teipsa. Riapi Sei! Ker. 12: 250-255).

Small, white-yellow embryogenic cultures of callus, grown on MVya50O mg/l of hygromycin, were regenerated by transferring to a pro-regeneration (RC) medium of 1-50 mg/l of hygromycin. PE medium consists of MV medium with 2 mg/l benzylaminopurine (BAP), 1 mg/l naphthalene acetic acid (MAA), and 5 mg/l abscisic acid (ABA). After 2 weeks of cultivation in the dark, the callus was transferred to the regeneration medium (CM). The composition of the CM medium is a MV medium with Zmg/l VAR and 0.5 mg/l MAA. Cultures of callus on and KM medium were incubated for 2 weeks at 282C under strong fluorescent light (325Kd). After 2 cm roots appeared in the rings, they were transferred to magenta boxes containing 1/2 M5 medium

IMygazpide apa 5Kood, 1962, A hemivey tedisht yog ghariy dgouli apa Bioazzauz m/yy (orasoo (izvtse siyyegev.

RNUsioIi. Riapi 15:473-497)| with 1/2 Vb-vitamins, 1Og/l of sucrose, 0.O5mg/l of MAA, 5Omg/l of hygromycin and 2.5g/l of -I deipye with pH of 5.8. Large plants of 8-15 cm with a well-developed root system were transferred to the ground (1

METKO-MIH: 1 top layer of soil) and grown in a greenhouse (29/24 day/night cycle, 50-60905 humidity, 12-hour photoperiodism). Rice plants were grown into mature transgenic plants. The seeds from these plants contained (9) genes inserted into rice cells, and when grown into plants expressed the proteins encoded by the inserted bu 20 dna.

Ko) Formula of the invention 1. A method of improving the nutritional profile of a plant, consisting of: - providing a DNA sequence encoding an enzyme capable of modifying the use of an intermediate substrate in the secondary metabolic pathway, related to the nutritional profile of a plant, and the said enzyme is not occurs in nature in the indicated secondary metabolic pathway; - transformation of a plant cell of the specified plant with an expression cassette containing the specified active DNA sequence operably connected to a seed-selective promoter, and - regeneration of a genetically modified plant from the specified plant cell, and the specified genetically modified plant is characterized by an improved nutritional profile in relation to the wild type of the specified plant plants 2. The method according to item 1, which differs in that the indicated secondary metabolic pathway is the phenylpropanoid pathway. 65 3. The method according to item 2, which differs in that the specified enzyme is a choline metabolizing enzyme. 4. The method according to item C, which differs in that the specified choline metabolizing enzyme is

Claims (1)

choline oxidase. 5. The method according to claim 4, which differs in that the specified expression cassette additionally contains a DNA sequence encoding betaine-aldehyde dehydrogenase capable of converting betaine aldehyde to betaine. 6. The method according to claim 2, which differs in that the specified enzyme affects ferulic acid. 7. The method according to claim 6, which differs in that the specified enzyme is ferulic acid decarboxylase. 8. The method according to claim 1, which differs in that the indicated secondary metabolic pathway is the secondary metabolic pathway of sugar alcohol. 9. The method according to claim 8, which is characterized by the fact that the specified substrate is selected from the group consisting of 7/0 Myoinositol, galactinol and UDP-derived sugars. 10. The method according to claim 8, which differs in that the specified substrate is myoinositol, and the specified enzyme is an enzyme acting on myoinositol. 11. The method according to claim 10, which differs in that the specified enzyme is myoinosit-O-methyltransferase. 12. The method according to claim 11, which differs in that the indicated myoinosit-O-methyltransferase has a DNA 7/5 sequence of myoinosit-O-methyltransferase Mezetbguapiletit stuviaipit. 13. The method according to claim 8, characterized in that said improved nutritional profile includes a reduced composition of phytic acid compared to the wild type of said plant. 14. The method according to claim 2, characterized in that said improved nutritional profile includes a reduced composition of sinapin compared to the wild type of said plant. 15. The method according to claim 2, characterized in that said improved nutritional profile includes an altered composition of lignin compared to the wild type of said plant. 16. The method according to any of items 1-15, which is characterized by the fact that the specified substrate is not a primary metabolite from the group consisting of glucose, amino acids, simple fatty acids and nucleotides. 17. The method according to any of items 1-15, which is characterized by the fact that the specified seed promoter is a phaseolin promoter or a lar/p promoter. 18. The method according to any of items 1-15, which additionally includes the following stage: (8) growing the above genetically modified plant under conditions that allow the formation of seeds and the regeneration of these seeds. 19. The method according to any of items 1-15, which is characterized by the fact that the plant is selected from dicotyledons or monocotyledons. 20. The method according to any of items 1-15, which is characterized by the fact that the above-mentioned plant is selected from the ise) family бБ/аз5з5/сасеае, preferably from the genus В/азз/са, preferably В/авв/са гара or Вгазв/ Jesus steamed. yu 21. The method of any one of claims 1-15, wherein said plant is selected from the group consisting of corn, canola, wheat, barley, alfalfa, soybeans and sorghum. at 22. A genetically modified plant or its offspring containing a recombinant DNA molecule stably integrated into the gene of the said plant, and the said recombinant DNA molecule contains: a seed-selective promoter; a DNA sequence operably linked to said seed-selective promoter, which encodes an enzyme capable of modifying the use of an intermediate substrate in a secondary metabolic pathway associated with the nutritional profile of the plant, and the enzyme does not occur naturally in said secondary metabolic pathway; and the mentioned genetically modified plant, its cell, seed or component of the specified genetically modified;" plant, or its offspring, is characterized by an improved nutritional profile of the plant in relation to the wild type of the specified plant. 23. A genetically modified plant or its offspring according to claim 22, which differs in that the indicated secondary -I metabolic pathway is the phenylpropanoid pathway. 24. A genetically modified plant or its offspring according to claim 23, which differs in that the specified enzyme is a choline metabolizing enzyme. c 25. A genetically modified plant or its offspring according to claim 24, which is characterized by the fact that the specified choline metabolizing enzyme is choline oxidase. me) 26. A genetically modified plant or its progeny according to claim 25, which is characterized by the fact that it additionally contains a Ge DNA sequence encoding a betaine-aldehyde dehydrogenase capable of converting betaine aldehyde to betaine. 27. A genetically modified plant or its offspring according to claim 23, which is characterized by the fact that the specified enzyme acts on ferulic acid. 28. A genetically modified plant or its progeny according to claim 27, which differs in that the indicated enzyme is ferulic acid decarboxylase. F) 29. A genetically modified plant or its offspring according to claim 22, which differs in that the indicated secondary metabolic pathway is the secondary metabolic pathway of sugar alcohol. 30. A genetically modified plant or its offspring according to claim 22, which is characterized by the fact that the specified substrate is selected from the group consisting of myoinositol, galactinol and UDP-derived sugars. 31. A genetically modified plant or its progeny according to claim 22, which differs in that the specified substrate is myoinositol, and the specified enzyme is an enzyme that acts on myoinositol. 32. A genetically modified plant or its offspring according to claim 31, which is characterized by the fact that the indicated enzyme is myoinosit-O-methyltransferase. 65 33. A genetically modified plant or its offspring according to claim 32, which is characterized by the fact that the specified myoinositol-O-methyltransferase has the DNA sequence of myoinositol-O-methyltransferase / ezeteguapilpetit stuviaspite. 34. The genetically modified plant or its progeny according to any of items 22-33, which is characterized in that the specified substrate is not a primary metabolite from the group consisting of glucose, amino acids, simple fatty acids and nucleotides. 35. The genetically modified plant or its progeny according to any of items 22-33, which is characterized in that the specified seed-selective promoter is a phaseolin promoter or a lar/p promoter. 36. The genetically modified plant or its progeny according to claim 22, characterized in that said improved nutritional profile includes a reduced phytic acid composition compared to the wild type of said plant. 70 37. A genetically modified plant or its progeny according to claim 22, which differs in that it includes a reduced composition of sinapin, compared to the wild type of the specified plant. 38. A genetically modified plant or its descendant according to claim 22, which differs in that it has a changed composition of lignin, compared to the wild type of the specified plant. 39. Genetically modified plant or its offspring according to any of items 22-38, which differs in that the /5 plant is selected from dicotyledons / monocotyledons. 40. A genetically modified plant or its progeny according to any of items 22-38, which differs in that the above-mentioned plant is selected from the family V/azz/saseze, preferably from the genus V/azvz/sa, preferably V/avv/sa gara or Vapor balance. 41. The genetically modified plant or progeny thereof according to any one of items 22-38, wherein said plant is selected from the group consisting of corn, canola, wheat, barley, alfalfa, soybeans, and sorghum. 42. Animal feed, which is at least partially obtained from a genetically modified plant or its offspring according to any of items 22-41, or from its cell, seed, or any other component thereof. with 6) with (Se) IF) IF) and - - and? -i 1 1 (o) Ko) ime) 60 b5