UA72185C2 - Cytochrome p450 monooxygenases - Google Patents

Abstract

Cytochrome 450II dependent monooxygenases and DNA molecules encoding these monooxygenases are provided, which are able to catalyze the biosynthethic conversion of aldoximes to nitrils and the conversion of said nitrils to the corresponding cyanohydrins, which are the presursors of cyanogenic glycosides. Moreover, the invention provides methods for obtaining DNA molecules according to the invention and methods for obtaining transgenic plants resistant to insects, acarids, or nematodes or plants with improved nutritive value.

Description

This invention relates to genetic engineering of plants using recombinant DNA technology in general, and, in particular, to enzymes involved in the biosynthesis of cyanogenic glycosides and genes encoding these enzymes. Proteins and genes according to the invention are used to increase the nutritional value or resistance of plants to parasites.

Cyanogenic glycosides are the basis of secondary plant metabolites in more than 2000 plant species. In some cases, they are a source of NCMs that can make the plant poisonous if consumed as food. For example, tubers of cyanogenic cassava (Mapipoi ezsshepia) are one of the most important food products in tropical areas. Cyanogenic glycosides present in the tubers can cause cyanide poisoning in humans due to insufficient processing of cassava products. Among other plant species, in which their potential toxicity when consumed excessively or used as animal feed is attributed to enzyme production

NSM - white clover (Tgitoiisht herep5), sorghum (Bogdpyt bBisoiog), flax (Sypyt i5naiizzitit), trigloquinin (Tgidiospip taghyita), lima beans (Rpazeoiy5 Ishpaii5), almonds (Atudaaits5) and apricot seeds (Rgypyv), cherries and apple trees (MaiYish5 ). Toxic properties can be reduced by blocking the biosynthesis of cyanogenic glycosides in these plants.

The circle of main precursors of natural cyanogenic glycosides is limited to five amino acids of hydrophobic proteins: valine, leucine, isoleucine, phenylalanine and tyrosine and one non-protein amino acid, cyclopentenylglycine. These amino acids are converted through a series of reactions to cyanohydrins, which are eventually linked to a sugar residue. Amygdalin, for example, is

O-p-gentiobioside, and prunazine - O-D-glucoside of (A)-mandelonitrile. Another example of cyanogenic glycosides having aromatic aglycons is the epimeric pair of cyanogenic glycosides durrin and taxiphyllin, which can be found in plants of the genera Zogdapyt and Tachy5, respectively. p-hydroxymandelonitrile, for example, is converted to durrin (D-ХО-glucopyranosyloxy-(5)-p-hydroxymandelonitrile) with the help of uridine diphosphate glucose (POPO) glycosyltransferase. It is believed that similar glycosyltransferases are present in most plants.

Vicianin and lucumin are also examples of amygdalin-like derivative disaccharides. Sambunigrin contains (5)-mandelonitrile as an aglycon and is therefore epimeric to prunazine.

Examples of cyanogenic glycosides having aliphatic aglycons are linamarin and lotaustralin, which are present in clover, flax, cassava, and beans. A detailed analysis of cyanogenic glycosides and their biosynthesis can be found in the work of Sopp, Mayshgmizzepzspayep 66:28-34, 1979, to which we refer.

The path of biosynthesis of cyanogenic glucoside of tyrosine derived from tyrosine has been widely studied (NaiKieg ei a), "Suapodepis diisozidev: (te Bryozupipeiiys raiiulau apa She epgute 5uziet ipmoimea": "Suapide sotroypav ip Bioodu", MMieu Spispevieg (Sira RGoshpdaiop Zutrozisht 140 pp. 49-66, 1988; NaiKieeg apa Moieg, Riapi

Rpuzioi 90:1552-1559, 1989; NaiKieg eiyi aiYi, Te 9. oi Vioi. Turn off 264:19487-19494, 1989; NaiKieeg apa Moieg,

Riapi Rnusioi. 96:10-17, 1990, NaiKieg apa Moiieg, TNe 9. oi Vioi. Spent 265:21114-21121, 1990; NaiKieg ei a),

Rgos. Mai). Asad si. BOTH 88:487-491, 1991; 5irrezep ei a!: "Viospetivzigu apa Viorpuvzisvo oi suyuspgote P450.

Zigisishge apa Rypsiip, Vioiesppoiodisa! Apa Esioides! Azresiv", Agospakou, A.I. (ed.), 1991, Kos ei ai, vii Ipi.

Sopi op Suyuspgote P450, Abrvigasi RII.053; apa 5irrezep ei ai, 8Ii Ipii. Sopi op Suyuspgote P450, Arvigasi

RIY.O16). I-tyrosine is converted to p-hydroxy-mandelonitrile (a precursor of durin) with M-hydroxytyrosine, M,M-dihydroxytyrosine, (E)- and (2)-p-hydroxyphenylacetaldehyde oxime and p-hydroxyphenylacetonitrile as intermediates. Two P450-type cytochrome monooxygenases are involved in this pathway. In cassava, a similar pathway involving cytochrome P450-dependent monooxygenase is used to synthesize linamarin and lotaustralin from valine and isoleucine, respectively (Kospei ai, Agspimez oi Viospetiziu apa Viorpuvisv, 292:141-150, 1992). The complex pathway from I-tyrosine to p-hydroxymandelonitrile in Zogdpyt bBisoiog showed that only two multifunctional cytochromes are required

P4A.50-dependent monooxygenases. The first enzyme, designated P4i5Otuv, converts tyrosine to p-hydroxyphenylacetaldehyde oxime. The second enzyme, designated P450ox, converts aldoxime to p-hydroxymandelonitrile. Given the similarity of the biosynthesis pathways of cyanogenic glucosides in different plants, we can draw a general conclusion that the above-mentioned pathway includes two multifunctional RA50-dependent monooxygenases, P450i and P4501i, which convert the previous amino acid into the corresponding aldoxime, and the aldoxime into the corresponding cyanohydrin, respectively. P.450i is a specific enzyme that determines the substrate specificity and, therefore, the type of glucoside produced, while less specificity is expected from P4A50i in the conversion of a number of structurally different aldoximes to the corresponding cyanohydrin.

Glucosinolates are hydrophilic, non-volatile thioglycosides that are found in several sequences of dicotyledonous angiosperms (Stgopdcib5i, "Te Emoishiop apa Siazviysayop oi Riozhegipd Riapiv", Mem Mogk

Voiapis! Sagaep, Vgoph, 1988). The simultaneous existence of cyanogenic glucosinolates and glucosides is mutually exclusive. The greatest economic importance of glucosinolates lies in their presence in all plants belonging to Vgazzisaseae (Sarragaie order5), numerous cultures of which have been used for centuries by mankind for seasonings, snacks, salads, as vegetables, as well as forage and fodder. Later, rapeseed (mainly Vgazzisa pari5 and Vgazzisa satrevigii) became the main commercial oilseed crop.

About 100 different glucosinolates are known, which have the same general structure, but differ in the nature of the side chains. Glucosinolates are formed from protein amino acids either directly or after one-time or multiple stretching of the chain (Opaegii! ei a!, Viospet. os. Zutr. 38:303-326, 1973). M-hydroxy amino acids and aldoximes, which have been identified as intermediates in the biosynthesis of cyanogenic glycosides, also serve as effective precursors for the biosynthesis of glucosinolates (Kipai ei a!, Rpuiospetivhigu 7:745-756, 1968; Maizytso ei ai!, Rpuospetivzigu 11:697- 701, 1972; Opaeetii, Yeshig. U. Viospet. 2:61-63, 1967). Cytochrome P4i50, used in the synthesis of cyanogenic glycoside, is thus functionally very similar to the corresponding biosynthetic enzyme in the synthesis of glucosinolate and therefore, most likely, belongs to the same family of P4A50 enzymes. Thus, the authors isolated a CcDNA clone from BZipari5 aira, which encodes the RA5O enzyme (ZEO IU MO:17) with 5495 identity with P45Otuv (SUR 79) and catalyzes the first step in the biosynthesis of glucosinolates, which is the formation of aldoxime from the original amino acid.

This cDNA clone shows approximately 9095 identity with the EbT sequence of Agiridorzi5 (T42902), strongly indicating that this cytochrome P150 enzyme is largely conserved in glucosinolate-containing species.

Reduction of the complex biosynthetic pathway for the above-described cyanohydrins to the catalytic activity of only two enzymes, cytochrome P4i50i and P.iB5Oi, allows controlling the biosynthesis of cyanogenic glycosides in plants. By transfection of gene sequences encoding one or both monooxygenases, the biosynthetic pathway for cyanogenic glucosides can be altered, restored, or initiated anew.

Alteration or introduction of the biosynthetic pathway for cyanogenic glycosides in plants by methods known to those skilled in the art is of great interest because the cyanogenic glycoside can be toxic to insects, acarids and nematodes. Therefore, altering, introducing or restoring the biosynthetic pathway for cyanogenic glycosides in plants or individual plant tissues makes the plants inedible to insects, mites or nematodes and thus helps to reduce crop damage caused by parasites. In combination with other insecticidal components, such as endotoxins Vasiy5 (pPyipdiep5z5), it is possible to achieve an even greater reduction of damage to the crop caused by parasites.

Alternatively, the gene sequences encoding the monooxygenases of the invention are used to construct DNA plasmids which, when transfected into plants such as cassava, sorghum or barley containing cyanogenic glycosides, remove the cyanogenic glycosides normally produced in the wild plants This can be achieved by expressing antisense or sense

RNA or ribozymes, as described in EP-458367-A1, EP-240208-A2, O5-5,231,020, UMO89/05852 and UMO90/11682, which inhibits the expression of monooxygenases according to the invention. This is very interesting because, despite numerous attempts, it has not yet been possible to completely remove cyanogenic glycosides from, for example, cassava or sorghum with traditional methods of growing plants. On the other hand, it was demonstrated that the increased number of cyanogenic glycosides in the epidermal cells of barley culture increases the sensitivity to the attacks of the causative agents of powdery mildew - fungi Eguzirpe dgatipi5 (Rogtopepvi, RIO peziz, soycipdep, 1989; Ibepipai! ei ai, Apdem/. Voi. 67: 97-106, 1993). A similar effect was observed in the cyanogenic rubber tree Nemea rgaziiepvzis after damage by the fungus Misgosusitsv shei (eregei ei ai, Riapi Rpuv5. 90:3-36, 1989) and in flax affected by SoPeioigispyt Iipi (I tsaikKe ei ai, Viospet. 7. 324:433 -442, 1953). In these examples, the quantitative resistance of the above and other plants where cyanogenic glycosides make the plant more susceptible to attack by microorganisms can be increased by preventing the production of cyanogenic glycosides in such plants. In barley, cyanogenic glycosides are located in epidermal cells. The expression of antisense, sense or ribozyme sequences is thus preferably, but not necessarily, stimulated by an epidermis-specific promoter.

The presence of cyanogenic glycosides in plants, even in small amounts, can also cause nutritional problems through the formation of unwanted carcinogens, as demonstrated in the example of barley. Barley malt, for example, contains small amounts of the cyanogenic glucoside epiheterodendrin, which in the process of producing alcohol from grain crops can turn into ethyl carbamate, which is considered a carcinogen. Recently, trial experiments have been conducted to limit the maximum permissible concentration of ethyl carbamate in food products, beverages and alcohols obtained by fermentation (Rosa Spetisa! Memu5 29:33.35, 1988).

UM0O95/16041 describes a DNA molecule encoding cytochrome P4i50I-monooxygenase, which catalyzes the conversion of an amino acid into the corresponding M-hydroxyamino acid, M,M-dihydroxyamino acid and the conversion of M,M-dihydroxyamino acid into the corresponding aldoxime. The starting amino acid is selected from the group consisting of tyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine and cyclopentenylglycine. DNA molecules correspond to either natural genes or their functional homologues that have arisen as a result of mutation, deletion, truncation, etc., but continue to encode cytochrome

P4A50i-monooxygenase, capable of catalyzing more than one reaction of the path of biosynthesis of cyanogenic glycosides.

Monooxygenases optimally contain a single catalytic center.

In addition, M/O95/16041 describes DNA molecules encoding cytochrome P450i1-monooxygenases, such as

Rab5bOoh 5ogdpyt bBisoiog() Moepsip. They catalyze the conversion of an aldoxime into a nitrile and the conversion of a nitrile into the corresponding cyanohydrin. The catalysis of the transformation of tyrosine into p-hydroxy-phenylacetonitrile by two multifunctional P.i50 enzymes explains why all intermediate products of this transformation, with the exception of (2)-p-hydroxyphenylacetaldoxime, are directed in one direction.

In the substrate of the algorithm proposed for the isolation of P45boh, the procedure for the isolation of RA5Otuv (SUR79, 5irrezep ei ai, Rgos. Mai. Asai si. OBA 91: 9740-9744, 1994) from sorghum is placed. With this approach, a diethylaminoethyl sepharose ion exchange column is used to bind RA50 enzymes, and the yellow pigments in the sample are not bound. Pigment removal serves a dual purpose. It is a prerequisite for the binding of PA450 enzymes to the following columns and allows to estimate the content of PA5O by spectrometry (binding of carbon monoxide and substrate). The present invention shows that P45boh, on the contrary, shows a low affinity for binding to a diethylaminoethyl sepharose column and is largely recovered in the running and washing fractions. To separate the activity of Ri45Ooch from yellow pigments using a phase based on triton X-114, a separation procedure is used. With optimal use of 0.6 to 1956 triton X-114, it is found that P450oh is distributed over both phases, unlike P45Otun, which is recovered in the detergent-rich upper phase. As the concentration of triton X-114 increases to 695, most of the P450ox is recovered from the detergent-poor lower phase, while the yellow pigments are in the upper phase. The disadvantage of using 695 Triton X-114 is the increased conversion of P450oh to its denatured P.4.20 form. This fact is used in the present invention for early stage purification of PA5O-monooxygenase, such as P4i45box, in order to clone genes encoding monooxygenases and stably transform a plant with genes encoding monooxygenase.

Isolation of P4i50ox and determination of partial amino acid sequences allows the construction of oligonucleotide probes and the isolation of cDNA encoding P45box. However, in this case, cloning was carried out according to a different principle.

The invention relates, first of all, to DNA molecules encoding cytochrome Ra5bi-monooxygenase, which catalyze the conversion of an aldoxime into a nitrile, and the conversion of the above-mentioned nitrile into the corresponding cyanohydrin. In an optimal variant, the aldoxime is a product of the transformation of an amino acid selected from the group consisting of tyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine and cyclopentenylglycine or an amino acid selected from the group consisting of I-tyrosine, I-valine and I1-isoleucine , catalyzed by P450I-monooxygenase, as described in M/O095/16041. The DNA molecules according to the invention correspond to either natural genes or their homologues that have arisen as a result of mutation, deletion, truncation, etc., but continue to encode cytochrome P.i5bi-monooxygenase, which catalyzes the conversion of an aldoxime into a nitrile and the subsequent conversion of the above-mentioned nitrile to the corresponding cyanohydrin. Monooxygenases according to the invention catalyze more than one reaction of the path of biosynthesis of cyanogenic glycosides and optimally contain a single catalytic center.

Cytochrome P4501 enzymes can be present in most living organisms. DNA molecules according to the present invention encoding Rai50i-monooxygenases are structurally and functionally similar to DNA molecules obtained from various plants that produce cyanogenic glycosides. In the optimal variant of the invention, DNA molecules are hybridized with a fragment of a DNA molecule with the nucleotide sequence presented in 5EO IU MO:1. The above-mentioned fragment has a length of more than 10 nucleotides, and in the optimal version - more than 15, 20, 25, 30 or 50 nucleotides. Factors that affect the stability of hybrids determine the strictness of observing the hybridization conditions and can be measured by the dependence on the melting temperature Tt of the hybrids formed. Calculations of Tt are described in many works. Example,

Koller with his co-authors (Keiiieg ei a!) describes in the work "OMA Rgorez: Vaskdgotspa, Arriisayop5, Rgosedigev",

MastiMyap Rybiiepeg5 ЦЯ, 1993, on pages 8 to 10, factors that should be taken into account when calculating values

Tt for hybridization reactions. DNA molecules according to the present invention are hybridized with the 5EO IU fragment

MO:1 at a temperature 30"C lower than the calculated Tt of the hybrid to be formed. In the optimal version, they are hybridized at temperatures 25, 20, 15, 10, or 57C lower than the calculated Tt.

For gene manipulation using recombinant DNA technology, the DNA molecule according to the invention, in addition to the gene encoding monooxygenase, may include DNA that allows replication and selection of DNA according to the invention in microorganisms such as E. soii, Vasiish5, Adhorasiegicht, Zigeryutusev or in yeast It may also include DNA that allows monooxygenase genes to be expressed and selected in homologous or heterologous plants. Such sequences include, among others, genes whose codon usage has been adapted to codon usage of heterologous plants, as described in M/O93/07278; genes that provide resistance to neomycin, kanamycin, methotrexate, hygromycin, bleomycin, streptomycin or gentamicin, to aminoethylcysteine, glyophosphate, sulfonylurea or phosphinothricin; to marker genes, such as galactosidase; to its natural promoter and transcription termination signals; to promoter elements, such as the 355 and 195 Samm promoters, or tissue-specific plant promoters, such as root-specific promoters (described, for example, in EP-452269-A2, UUOU1/13992, |I5-5,023,179), green leaves , such as phosphoenolpyruvate carboxylase (PERS) of maize, pith or pollen (described, for example, in

UU093/07278) or induced plant promoters (ER-332104); as well as to heterologous transcription termination signals.

The present invention also relates to P450y-monooxygenases, which catalyze the conversion of an aldoxime to a nitrile and the conversion of the aforementioned nitrile to the corresponding cyanohydrin. In the optimal version of the invention, monooxygenases are purified and used to create monoclonal or polyclonal antibodies that specifically bind to monooxygenases. In particular, cytochrome P45b0ox, which has a molecular weight of 55 KkDa, as determined by electrophoresis with sodium dodecyl sulfate in a polyacrylamide gel (5ZO5-RACE), is isolated from Khogapyt bBisoog (I) Moepsp. Its amino acid sequence is presented in ZEO IU MO:2.

The catalytic properties of P4a5O0oh resemble the activity of cytochrome P450, which is observed in microsomes from the rat liver (OeMavieg ei ai, U. Ogd. Spet. 5074-5075, 1992). A characteristic of cytochrome P45O0ox and other members of the cytochrome P45Oox family is that the dehydration of aldoxime to the corresponding nitrile depends on the presence of reduced nicotinamide adenine dinucleotide phosphate (MAORN), but this dependence can be overcome in some cases by adding sodium dithionite or other reducing agents.

Of all the known sequences for cytochrome RA4A5O0 enzymes, cytochrome P450ox shows the highest amino acid sequence identity (4495) to the avocado enzyme СУР7ТА1Т and less than 4095 identity to all other members of the СУР71 family. Avocados do not produce cyanogenic glycosides, and SUR71A1 does not catalyze the conversion of an aldoxime into a nitrile and the conversion of the aforementioned nitrile into the corresponding cyanohydrin. Thus, according to the present invention, a family of cytochrome P5O monooxygenases can be defined, members of which catalyze the conversion of aldoxime to the corresponding cyanohydrin and have 4095 or higher amino acid sequence identity to the sequence of cytochrome P45boh. In an optimal variant, the identity of the amino acid sequence to cytochrome Ri45boh is higher than 5095 or higher than 5595.

P450oh was proposed to be designated as the first member of the new SUR71 subfamily (SUR71E1), as it is grouped with other SUR71 sequences in dendrograms that show the order of sequences in a graphical way. In general, according to the provisions of the nomenclature committee, a cytochrome P450, in order to be assigned to a new SUR family, must have less than 4095 sequence identity at the amino acid level, and sequences that are more than 5595 identical are assigned to a single subfamily .

When determining the order of sequences, not only the identity of the sequences is considered, but also the similarity of the sequences, for example, the same overall charge or similar hydrophobicity/hydrophilicity of individual amino acids. In this order, P45Oox clusters with other SMU7 sequences and thus should be included in the SMU71 family, despite the fact that it shows less than 4095 identity to all other members of the SMU71 family, with the exception of SMU7TA71 from avocado. Because it shows low sequence identity to other members of the family, it should be assigned to a new subfamily. All other members of the SUR71 family belong to non-cyanogenic species and their function is unknown. The catalytic properties of previously identified P450 belonging to the CMP71 family remain unclear. They are believed to be involved in the hydroxylation of terpenes. None of them are mentioned to use oximes as substrates, or to be multifunctional and convert aldoximes to nitriles and cyanohydrins.

Another version of the embodiment of this invention is related to the method of preparation of cDNA encoding cytochrome

RabBO-monooxygenase, which catalyzes the conversion of an aldoxime into a nitrile and the conversion of the aforementioned nitrile into the corresponding cyanohydrin. It includes

(a) isolation and solubilization of microsomes from plant tissue that produce cyanogenic glycoside, (b) purification of cytochrome P450 monooxygenase, (c) production of antibodies against purified monooxygenase, (c) probing the cDNA expression library of plant tissue that produces cyanogenic glycoside, with the aforementioned antibody, and (e) selection of clones that express monooxygenase.

Microsomes can be isolated from plant tissues that show high activity of the enzymatic system responsible for the biosynthesis of cyanogenic glycoside. These tissues can be different in different plant species. The optimal source of microsomes is freshly isolated sprouts collected within 1 to 20 days, preferably 2 to 10 days, and best 2 to 4 days after germination. Etiolated seedlings of plants that produce cyanogenic glycosides are considered the best, but seedlings germinated in the light can also be used. After isolation, the microsomes are dissolved in a buffer containing one or more detergents.

Optimal detergents are KEMEH 690 (9. Gogepigep A/5, Kmizidaga, Oeptagk), reduced Triton X-100 (ATX-100), Triton X-114 and SNAR5.

Cytochrome P. and 50-monooxygenases can be purified using standard protein purification technologies, such as ultracentrifugation, fractional precipitation, dialysis, electrophoresis with sodium dodecyl sulfate in a polyacrylamide gel (5O5-RACE), and column chromatography. Other possible columns include ion-exchange columns such as diethylaminoethyl sepharose (DEAE), reagent columns such as the Sirasgop UePom/3 agarose column, the Sirasgop Rise agarose column and the Keasiime Gay 120 agarose column, and filtration columns such as like a column with Zerpasgu brand gel! 5-1000.

The content of cytochrome P450 in individual fractions is determined by the difference in the spectra of carbon monoxide. A particular problem during the isolation of P450ox, which also makes it difficult to determine the amount of P45Oox, is its mutual mixing with yellow pigments during the initial stages of purification instead of binding on an ion exchange column, which is usually used for the purification of P450-enzymes, for example, P45Otuv. The presence of yellow pigments prevents P45b0oh from binding to many different materials on the column and, thus, represents a major obstacle to further purification. However, the separation of P4i5Oox from yellow pigments can be carried out at a temperature that causes phase separation of triton X-114. This method was optimized for P450ox recovery and pigment removal by increasing the amount of Triton X-114. At 69, which is six to ten times higher than the level used for other P4A50s, approximately 8095 of the active part of P450ox reaches the clear lower phase. In addition to the removal of yellow pigments, it is almost impossible to achieve greater purification at this stage. However, when using P45boh, which contains the lower phase, on a Sirasgop Brisé column, gradient elution of the salt gave almost homogeneous RaBboh, as evidenced by the presence of a major Coomassie-stained band with an apparent molecular weight of 55 kDa in those fractions that, upon reduction, showed RA5Oox activity.

The isolated P450ox gave a spectrum of carbon monoxide with an absorption peak at 450 nm, but a relatively large part of the isolated enzyme was present in the form of denatured P420. Continuous transformation

Ра4б5Ох on the denatured form of Р4и20 interfered with the quantitative determination of the total content and specific activity of Р45Ох at different stages of the isolation process. In addition, the specific activity of P450ox depends on the inhibitory effect exerted by the various detergents used. The total content of RA5BO in the fractions, thus, should be considered determined semi-quantitatively.

Purified proteins are used to detect antibodies, for example, in mice, goats, rams, rabbits or chickens after administration. From 5 to 50 mcg of protein is administered several times over approximately 14-day periods.

In the optimal version of the invention, from 10 to 20 Ωkg are injected from 2 to b times during 14-day periods, the injections are carried out in the presence or absence of adjuvants. Immunoglobulins are purified from antisera and the spleen is used for hybrid fusion as described in Napom apa Gape, "Apiirodie5: A

Iarogayugu Mapia!", Soya Brgypd Nagbrog I arogayogu, Soyy brgipd Nagbrog, MU, 1988, to which the authors refer. Antibodies that specifically bind to cytochrome Ra5bi-monooxygenase are also used in plant breeding to detect plants that have altered cytochrome P450-monooxygenases, and therefore cyanogenic glycosides, are produced in quantities.

These methods of preparation of cDNA libraries of plant tissues are widely described in the work of Zatogook ei ai,

Moiesshiag siopipd: And Iarogaigu tapiai!. Soya 5rgipd Nagrog IGarogayugu Rgez5, Soy Zrgipd Nagbrog, MU, 1989, to the essential part of which, which concerns the libraries of KDNK, the authors make a reference. PolyA" RNA is isolated from plant tissue that exhibits a high activity of the enzymatic system responsible for the biosynthesis of cyanogenic glycosides. These tissues can be different in different plant species. The optimal tissue for isolation of polyA" RNA is tissue from freshly isolated sprouts collected within 1 up to 20 days, better - from 2 to 10 days and best - from 2 to 4 days after germination. The obtained cDNA libraries are probed with antibodies that specifically bind cytochrome P4501i-monooxygenase, and clones expressing monooxygenase are isolated.

An alternative method of preparing cDNA encoding cytochrome P4A501i-monooxygenase includes (a) isolation and dissolution of microsomes from plant tissue that produces cyanogenic glycosides, (b) purification of cytochrome P450i-monooxygenase, (c) obtaining the complete or partial protein sequence of monooxygenase, ( in the construction of oligonucleotides that determine the DNA encoding from 4 to 15 amino acids of the above-mentioned monooxygenase protein sequence (e) probing cDNA libraries of plant tissue that produces cyanogenic glycoside with the above-mentioned oligonucleotides, or DNA molecules obtained from the amplification of cDNA using the polymerase chain reaction ( RSK) using the aforementioned oligonucleotides, and (is) selection of clones that encode cytochrome P450y-monooxygenase.

The amino acid sequences of internal peptides resulting from protease cleavage can be obtained by standard methods such as Edman cleavage. Oligonucleotides, which determine DNA encoding partial protein sequences of monooxygenase according to the invention, are obtained by reverse translation of parts of the protein sequence according to the genetic code.

Protein sequences that are encoded by DNA sequences of low degeneracy are optimal for reverse translation. They have a length of 4 to 15, preferably 5 to 10 amino acids. If necessary, the codons used in the oligonucleotides can be adapted to use codons of plant origin (Migtgau et al, Misieis Asia5 Kezeagsp 17:477-498, 1989). The obtained oligonucleotides are used for probing the cDNA library, as described in the work of Zatgook ei ai, (MoiIesshiag siopipd: A

Iarogayugu tapiai. Soya brgipd Nagbrog I aroghaigu Rgez5, Soya brgipd Nagrog, MU, 1989) for clones that are capable of base pairing with the aforementioned oligonucleotides. Alternatively, oligonucleotides are used in a polymerase chain reaction, the methodology of which is known to specialists, with plant cDNA as a matrix for amplification. In this case, the obtained amplification products are used for probing libraries

KDNK. Clones encoding cytochrome P4501-monooxygenase are isolated.

An alternative method of gene cloning is based on the creation of a gene library consisting of expression vectors. Thus, similar to the methods described above, genomic DNA, preferably cDNA, is first isolated from cells or tissues capable of expressing P4A501-monooxygenase, and then spliced into the appropriate expression vector. Gene libraries formed in this way can then be subjected to screening using appropriate means, ideally antibodies. Clones that include the desired gene or at least part of the gene as an insert are selected.

Alternatively, cDNA molecules encoding cytochrome RaB5O-monooxygenase, which catalyzes the conversion of an aldoxime to a nitrile and the conversion of the aforementioned nitrile to the corresponding cyanohydrin, can be obtained by (a) creating degenerate oligonucleotides spanning from 3 to 10 amino acids of conserved regions of cytochromes A- type, (b) use of degenerate oligonucleotides to amplify one or more cytochrome-specific DNA fragments using the polymerase chain reaction, (c) screening of a cDNA library using cytochrome-specific fragments to obtain full-length cDNA, (d) expression of full-length cDNA in microbes, (e) identification of hosts expressing cytochrome P450 monooxygenase, which catalyzes the conversion of an aldoxime to a nitrile and the conversion of the aforementioned nitrile to the corresponding cyanohydrin, and (e) purification of the cloned DNA of the aforementioned host.

Complete DNA from a DNA library, ideally from a cDNA library, is used as a template in the polymerase chain reaction (PCR) with one or more primers representing conserved regions of A-type cytochromes (YuOig5i ei ai, Ogid Meiaroizt apa Ogid IpbFegasiop5 12: 189-206, 1995), which are considered to be derived from a common ancestor - plant cytochrome P4i50. Based on the order of cytochrome P450 A-type sequences, three well-conserved regions at the amino acid level can be identified: region 1 (MOKEX(/AU)K, region 2 EHRERKE, and region C REOHOKEXHSO. Primers containing degenerate inosine (I) can be designed. , each of which includes from C to 10, in the optimal version - about 5 or 6 amino acids of the two corresponding regions. Polymerase chain reaction (PCR), for example, is carried out in three consecutive stages. Stage 1 using a primer that includes a common region of EHREKE, and a standard T/ primer, which includes the T7 promoter in the library vector, amplifies cDNAs derived from mRNAs encoding cytochrome P450-type A. A second step polymerase chain reaction (PCR) using primers that include two shared regions and amplified DNA from step 1 as a matrix, in the optimal version, amplifies a 100-bp fragment, which is then ligated into rVinezsghir and sequenced. Gene-specific primers are created on the basis of oder wife

DNA sequence. they are used in step C in combination with a primer complementary to the poly-A tail (primer YAT-M) and tolymerase chain reaction (TCR) DNA from step 1 as a template for amplification, a DNA fragment of approximately 500 bp, which is used as a gene-specific probe for isolation of full-length cDNA. Such a polymerase chain reaction is characteristic not only for isolation

P4a5BoOkh, and is common for the isolation of cytochrome P450 A-type. The resulting P450 A-type cytochromes must be heterologously expressed to determine their function.

cDNA clones or polymerase chain reaction (PCR) products prepared as described above, or fragments thereof, can be used as hybridization probes in the process of identifying further DNA sequences encoding a protein product that exhibits P450ii-monooxygenase activity from a homologous or heterologous organic source. such as fungi or heterologous plants.

A suitable source is tissue from plants containing cyanogenic glycosides.

The aforementioned clones or polymerase chain reaction (PCR) products can also be used as restriction fragment polymorphism (RRP) markers to determine, for example, the location of a cytochrome P450-monooxygenase gene or a closely related feature in the plant genome, or for marker breeding (ER-A 306139; UMO89/07647I.

Using the methods described above, it is possible to isolate various genes that encode P4501i-monooxygenase.

The above-mentioned genes are used according to the method of producing purified recombinant cytochrome

RabO-monooxygenase, which catalyzes the conversion of an aldoxime into a nitrile and the conversion of the aforementioned nitrile into the corresponding cyanohydrin; the method comprises (a) constructing a gene encoding the aforementioned monooxygenase to be expressed in a host organism such as bacteria, yeast or insect cells, (b) transforming the aforementioned host organism with the engineered gene, and (c) release of protein from the host organism or culture supernatant.

In the optimal version of the invention, this method is used to obtain purified recombinant cytochrome P45boh or cytochrome P45O0oh, which has been modified by traditional methods of gene technology. In the optimal variant, the changes lead to enhanced expression of the recombinant protein or to altered substrate specificity.

DNA molecules according to the invention are used to obtain transgenic plants resistant to insects or mites. Specific examples, which are not exhaustive, are given in Table B MUO95/16041 (page 45), as are the nematodes described below. For convenience, only the aforementioned Table is not repeated in this description, but it is mentioned by the authors by reference to the description from M/095/16041. In the optimal variant, the transgenic plants are resistant to SoiIeoriega and I eridoriega, such as the western long-bearded bug (Oiabgoiisa migditega migditega), the northern long-bearded bug (Oiargoiisa Iopdisogpi5 ragregi), the southern long-bearded bug (Oiabgoiisa ipadesitripsiaia Ppomagii), the cotton bollworm, the corn borer, , a pink boxworm and a scoop.

Nematodes are major animal parasites of plants, causing losses to agriculture worldwide estimated at 5100 billion each year. Some nematodes cause changes in plant cells at the feeding sites and feed in one place for several hours or much longer. Among them are species belonging to the genera MeiIoidodupe Siorodega, Neiegodega, Koiuiepspii5, Tuiepspii5, Massoriv,

Khirpipeta, I opdidogiv, Ragaiopdidogiv, Sturpodega; TgornoiyuIeppstsiv, Netisusiiorpoga, StisopeteiPa, Megpiiv and Neiiiosoiuiepspi5. Genera that feed for a more limited time in one place include

RgaiuIepspiv, Nayorpoiiv5, Nigzsptappieia, Tispodogiv, Ragaiisnpodogiz, Oiuiepspyv5, ArPpeiIepspoidev,

ZsssheiPopeta and VeiIopoiativ5.

The transgenic plants include DNA encoding novel monooxygenases that catalyze the conversion of the aforementioned aldoxime to a nitrile and the conversion of the aforementioned nitrile to the corresponding cyanohydrin. In addition, transgenic plants may include monooxygenase genes genetically linked to herbicide resistance genes. Transgenic plants are optimally monocotyledonous or dicotyledonous plants. Specific options are given in Table A of UUO95/16041 (pages 33-44). For convenience, only the aforementioned Table is not repeated in this description, but it is mentioned by the authors with reference to the description from M/095/16041. In the optimal version, they are selected from the group consisting of corn, rice, wheat, barley, sorghum, cotton, soybean, sunflower, cereal grasses, oilseed rape, sugar beet, broccoli, cauliflower, cabbage, cucumbers, sweet corn, radish daikon, turnips, lettuce, melon, pepper, pumpkin, tomatoes and watermelon.

Plants are produced by a method that includes (a) introducing into a plant cell or plant tissue that can be regenerated into a whole plant,

DNA including a gene that can be expressed in this plant encoding a monooxygenase according to the invention, (6) selection of transgenic plants.

Similarly, DNA molecules according to the invention are used to obtain transgenic plants expressing antisense or sense RNAs or ribozymes intended for genes of endogenous РА50О monooxygenase. The expression of these molecules in transgenic plants weakens the expression of cytochrome P4A50y monooxygenase. Such plants exhibit improved disease resistance or nutritional value due to reduced expression of cyanogenic glycosides. Such plants can be produced by a method which includes (a) introducing into a plant cell or tissue, which can be regenerated into a whole plant, DNA encoding a sense RNA, an antisense RNA, or a ribozyme, the expression of which attenuates the expression of cytochrome

P45O-monooxygenase, and (6) selection of transgenic plants.

There are many effective ways of introducing DNA into plant cells, which are based on the use of gene transfer vectors or direct gene transfer methods.

According to one of the possible methods of insertion of the gene sequence into the cell, infection of the plant cell with the bacterium Adgorasieegst isteiasiep5 and/or Adgorasieegist gpigodepe5, which was transformed by the above-mentioned gene sequence, is used. The transgenic plant cells are then cultivated in a suitable culture medium known to specialists to form sprouts and roots from them, and eventually the whole plant.

The present invention relates to the so-called conversion of sheet discs using Adgobasiegiit (Nogiesp eyi aiYi, Zsiepse 227:1229-1231, 1985). Sterile leaf disks from suitable plants are incubated with cells of Adgobrasiegisht, including one of the chimeric gene sequences according to the invention, and then transferred to or on a suitable nutrient medium. Especially suitable, and therefore optimal within the scope of this invention, are I 5-mediums, solidified after adding agar and enriched with one or more traditional plant growth regulators, mainly selected from the group of auxins consisting of snaphthylacetic acid, picloram, 2, 4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-butyric acid, indole-3-lactic acid, indole-3-succinic acid, indole-3-acetic acid and p-chlorophenoxyacetic acid, and from the group cytokinins, consisting of kinetin, b-benzyladenine, 2-isopentenyladenine and zeatin. The optimal concentration of auxins and cytokinins is from 0.1 mg/l to 10 mg/l.

After incubation for several days, optimally - after incubation for 2-3 days at a temperature from 207"C to 40"C, better - from 23"C to 35"C and best of all - at 25"C and with diffuse lighting the discs are transferred to a suitable medium for germination. I 5 medium, which does not contain auxin but contains cytokinin and to which a selective substance has been added, is especially suitable for selecting transformants. The cultures are kept in the light and transferred to a new medium at appropriate intervals, in the optimal option - at intervals of one week.The developing green sprouts are cut off and cultivated further in a medium that causes the sprouts to form roots.

Particular preference according to the present invention is given to the IZ-medium, which does not contain auxin or cytokinin, but to which a selective substance has been added for the selection of transformants.

In addition to mediated Adgobrasiegisht transformation, according to this invention, it is possible to use direct transformation methods for inserting the gene sequence according to the invention into plant material.

For example, the genetic material contained in the vector can be inserted directly into the plant cell, for example, using purely physical procedures, for example, by microinjection using fine-drawing micropipettes (Meshipatz ei ai, Tpeogeiisa! apa Arrieiei Sepeiiisv 74:363 -373, 1987), electroporation (OS'Naiship ei ai, Tpe Riapi Seii! 4:1495-1505, 1992; УМО92/09696) or, in the optimal version, bombardment of cells with microparticles covered with transforming DNA (MisgorgoyesshShe

Votbragatepi"; Uapd ei a. Riapi MoIescag Vioidou 11:433-439, 1988; Sogaop-Katt ei ai, TNe Riapi Seii! 2:603- 618, 1990; MeSare ei ai, Vio/ThesppoIodu 11:596-598, 1993 ; Spgizoioi ei, Riapi Rpuzioi!. 87:671-674, 1988; Koliye!ei ai, Vioitesppoiodou 11: 194-200, 1993).In addition, the plant material to be transformed,

optionally pretreated with an osmotically active substance such as sucrose, sorbitol, polyethylene glycol, glucose, or mannitol.

Among other possible methods of direct transfer of genetic material into a plant cell is processing of protoplasts using procedures that change the plasma membrane, for example, treatment with polyethylene glycol, heat treatment or electroporation, or a combination of these procedures (ZPSHNO ei ai,

Phytochemistry 3:1099-1103, 1985).

Another method of direct introduction of genetic material into plant cells, which is based on purely chemical procedures and which allows transformation to be carried out very efficiently and quickly, is described in the work

Medgyi ei a, Riapi MoiIesshiag Vioiodu 8:363-373, 1987.

Direct gene transfer using cotransformation is also suitable for the transformation of plant material (Zspospeg ei ai, Viotesppoiodu 4:1093-1096, 1986).

The list of possible methods of conversion given above as an example does not pretend to be complete and by no means exhausts the subject of the invention.

The genetic properties described above, engineered into transgenic seeds and plants, are transmitted through sexual reproduction or vegetative growth and can thus be preserved and passed on to offspring. In general, the aforementioned conservation and transmission use traditional agricultural techniques developed specifically for specific purposes, such as ploughing, sowing or harvesting. Special technologies such as hydroponics or growing in greenhouses can also be used. Since cultivated crops are vulnerable to attacks and damage caused by insects or infections, as well as to weeds, measures are taken to control weeds, plant diseases, insects, nematodes and other negative factors in order to improve yields. These include mechanical measures such as tillage or removal of weeds and infected plants, as well as the use of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulators, ripening agents and insecticides.

The beneficial genetic properties of transgenic plants and seeds according to the invention can also be used in plant breeding, which aims to develop plants with improved properties such as resistance to parasites, herbicides or stresses, increased nutritional value, increased yield or improved structure that reduces losses from lying down or shedding. Different stages of breeding are characterized by well-defined human intervention, such as the selection of lines for crossing, the direction of pollination of parental lines or the selection of suitable offspring. Depending on the required properties, different cultivation measures are used. Such techniques are well known to those skilled in the art and include, but are not limited to, hybridization, inbreeding, backcrossing, multiline breeding, interbreeding, interspecific crossing, aneuploidy techniques, etc. Hybridization methods also include sterilizing plants to produce male or female sterile plants by mechanical, chemical, or biochemical means. Cross-pollination of male-sterile plants with pollen from different lines ensures that the genome of male-sterile and female-fertile plants equally acquires the properties of both parental lines. Thus, transgenic seeds and plants according to the invention are used to grow improved plant lines, which, for example, increase the efficiency of traditional methods, such as treatment with herbicides or pesticides, or allow to do without the aforementioned methods thanks to their changed genetic properties. As an alternative, new crops with improved resistance to stress can be obtained, which, thanks to their optimized genetic "equipment", give a harvest of better quality than products that are not able to tolerate adverse conditions of development.

In seed production, the quality and uniformity of seed germination are essential product characteristics, while the quality and uniformity of germination of seeds collected and sold by farmers are irrelevant. Because it is difficult to keep the crop separate from other crops and weed seeds, to control seed diseases, and to obtain seeds with good germination, fairly broad and well-defined methods of seed production have been developed by growers experienced in the cultivation, storage, and sale of pure seed. Thus, it is common among farmers to purchase certified seed that meets specific quality standards, instead of using seed collected from their own harvest.

Breeding material to be used as seed is usually treated with a protective coating that includes herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof. Traditionally used protective coatings include compounds such as captan, carboxin, thiram (TM), metalaxyl (Argop"), and pyrimiphosmethyl (Acylin). If desired, these compounds are formulated with other carriers, surfactants, or lightening adjuvants. coatings traditionally used in development to provide protection against damage caused by bacteria, fungi and animal parasites. Protective coatings are applied by impregnating the dilution material with a liquid formulation or by coating with a combined wet or dry formulation. Other applications are possible, such as processing aimed at sprouts or fruits.

Another aspect of the present invention is to provide new agricultural methods, such as the above methods, which are characterized by the use of transgenic plants, transgenic plant material or transgenic seeds according to the present invention.

The following examples further describe the materials and methods used in the practice of the invention and the consequences of such use. They are offered for illustration and their listing should not be considered exhaustive of the claimed invention.

Examples

Example 1: Preparation of microsomes

All steps related to the preparation of microsomes are carried out at 4"C, unless otherwise indicated. All buffers are degassed with mixing and masso and purged with argon. Seeds of Zogapyt bBisoiog (I) Moepsi (hybrid

З51000 from AdgiRgo, Texas, USA) germinate in the dark for 4 hours. at 28"C on metal sieves covered with gauze. Microsomes are prepared from approximately 3 cm long etiolated seedlings. Seedlings are collected and homogenized using a mortar and pestle in 2 volumes (vol/weight) of 250 mM sucrose, 100 mM Tgtgisipe (pH 7 ,9), 2mM ethylenediaminetetraacetic acid (EDTA) and 2mM dithiothreitol (DTT). Privinylpolypyrrolidone (0.1g/g crude mass) is added before homogenization. The homogenate is filtered through a 22μm nylon cloth and centrifuged for 10 minutes at 16,500 x 9. The supernatant is centrifuged for 1 hour at 165,000 x d. The microsomal pellet is resuspended and homogenized in isolation buffer using a Reutzeg-EiMepiegp homogenizer with a Teflon pestle. After repeated centrifugation and repeated homogenization, the homogenate is frozen in liquid nitrogen and stored at -807C until use.

Example 2: Enzyme analyses: Determination of the total amount of cytochrome P450. Determination of the total amount of cytochrome P4A50 is carried out by spectroscopy using the extinction coefficient of 91MM' 1tst" for the adduct between reduced cytochrome P450 and carbon monoxide (A450-490) (Otyga e6i ai, U.

Axes Spent 239:2370-2378, 1964).

Example 3: Purification of cytochrome P45boh All steps related to the purification of the enzyme are carried out at 4"C, unless otherwise indicated.

Buffer A: 8.990 glycerol 10 mM KHROZ/KhNRO: (pH 7.9) 0.2MM EDTA 2.0mM TT 1.095 (v/v) Kepeh 690 0.0595 ATX-100

Buffer C: 8.990 glycerol 40MM KHROZ/K»HRO:X (pH 7.9) 5.0MM EDTA 2.0mM TT 1.095 (mass/volume) SNAR5

Buffers are degassed three times with mass mixing before detergent and OTT are added. Between degassings, the buffer is purged with argon. The ability of different column fractions to metabolize radiolabeled p-hydroxyphenylacetaldoxime is monitored throughout the purification process to detect the presence

R4AiBOoh in fractions.

Microsomes (400 mg of protein in 20 ml) are diluted to 100 ml with a buffer consisting of 8.995 glycerol, 10 mM KHROZK»HRO:x (pH 7.9), 0.2 mM EDTA, 2 mM OTT, after which, with constant stirring, slowly add 100 ml of 10 mM KHROK» HRO" (pH 7.9), 8.995 glycerol, 02 mM EDTA, 2 mM OTT, 0.195 ETH-100 (v/v), 295 keg. After further stirring during ZOkhv. and further ultracentrifugation At 150,000 rpm for 35 minutes, approximately 190 ml of the supernatant is applied at a flow rate of 100 ml/h on a 5x5 cm EE/5-100 column with diethylaminoethyl sepharose (20/80 raw volume,

RPpagtasia) equilibrated in buffer A. Diethylaminoethyl sepharose ion exchange resin is diluted with 5-100-zerpagose gel filtration material in a ratio of 1:4 to avoid excessive concentrations of cytochrome P450 enzymes during binding, which can lead to irreversible aggregation. The column is then washed with 150 ml of buffer A. P450oh binds weakly to the column and is separated mainly in the washing and washing fractions, which contain a yellow pigment. Combine fractions (approximately 200ml) containing P45Oox, identified by their absorbance at 420 nm, their CO binding spectra, and their ability to metabolize the oxime in reduction experiments (see Example 4). They are used for further cleaning or frozen.

In the mixed fractions of P450oh, during constant stirring, a mixture of glycerin and Triton X-114 is added dropwise in the appropriate amount until the concentration of 30905 (vol/volume) of glycerin and 695 T/op X-114 is reached by adding dropwise in the appropriate amounts of the mixture glycerin and Tgyop X-114. Mixing continues for 20 minutes, after which centrifugation is carried out for 25 minutes at 24,500 x 9, 2570 (the temperature at which the phase distribution of Tgyop X-114 occurs) without stops. Two phases are formed: a yellow upper phase and a transparent lower phase. The lower phase, which contains the main part of cytochrome activity

P4a5BoOkh, diluted 2.5 times to approximately C50ml with buffer C and applied at a flow rate of 7Oml/h to a 1.9x5cm column with agarose of the Sirasgogiritsche ZSA brand, equilibrated in buffer C. The column is washed with 50ml of buffer C and cytochrome P45boh, which remains, is subjected to elution of approximately bOml of a linear gradient of 0-1.5M

KSI in buffer C. Ten fractions, which during the electrophoresis of sodium dodecyl sulfate in a polyacrylamide gel (505-RACE) reveal the presence of a single polypeptide band in the area of 50-60 kDa, are mixed and dialyzed in a nitrogen atmosphere for 24 hours against 1 l of 8.995 glycerol, 10 MM КНгРО/К» HRO: (pH 7.9), 5 mM EDTA, 2 mM

OT (dialysis buffer) to reduce the content of salts and detergents. The enzyme preparation is frozen in liquid nitrogen and stored at -807C.

Example 4: Determination of the characteristics of cytochrome P45boh obtained by isolation from sorghum microsomes 4.1. Determination of molecular weight and amino acid sequence

The molecular weight of P45boh, as determined by electrophoresis with sodium dodecyl sulfate in a polyacrylamide gel (50O5-RACE), is 55 kDa. The protein band, which corresponds to P450oh, isolated from a column with agarose of the Sirasgop risse ZA brand, is cut out of 8-2595 5O5-polyacrylamide gel and subjected to electroelution. The electroeluted protein is treated with endoproteinase SISH-C (protease sequencing

M8, 18h, 23") according to the manufacturer's instructions (Voephipdeg Mapppeit) using an approximate 1:100 mass ratio between proteinase and protein. The electroeluted protein and a sample of the treated protein are subjected to 50O5-RACE, and the protein and fragments are transferred to PgoVioi membranes (Arriei Viozuzziet5 ).Coomassie-stained sections of the membrane are excised and subjected to M-terminal amino acid sequencing on an Arriea Viosuziet5 sequencer, model 470A, equipped with a system phenylthiohydantoin amino acid analyzer, model 120A.

M-terminal amino acid sequencing yielded two sequences that could be read independently of each other due to their difference in relative content. Search using the database (VI AB5T) showed that: the sequence -5YI MKESMOMEESTI. differs in only one position from the M-terminal sequence of B-

subunits of ATRAse of barley vacuoles (Nogdent mtsIdage) which is MY MKESAOMEESTS. (inventory number I11862). The B-subunit of barley has a molecular mass that corresponds to the theoretical - 54 kDa (20). The presence of the B-subunit of ATPase of vacuoles as a contaminating impurity in the P45boh preparation was detected by Western blotting, which showed a single band of 55 kDa when using a monoclonal antibody produced against the B-subunit of ATPase of vacuoles from oat roots (Og. Nemep 52ee). The B-subunit can be removed from the P4i45boh preparation by immobilization on antibody-coated microtiter wells. This approach allows you to definitely determine M - the final amino acid sequence P4А5б0ох as -

ATTATROG /ООБМРЕО and, in addition, gives the sequence of the peptide fragment of internal PaА5Ох,

MORGMAOGOKADAA. Attempts to remove the residual amount of the B-subunit of ATPase from the vacuoles led to the formation of carbon monoxide difference spectra, in which the 420 nm component, representing the inactive denatured form of P420 of cytochrome P4i5boh, increased significantly, and to the loss or significant reduction of the ability to restore the activity of Pi45boh in the obtained fractions. This reflects the instability characteristic of P45Ooh. The B-subunit of ATPase of vacuoles obviously does not have any catalytic properties associated with P45boh.

Thus, the presence of the B-subunit as a contaminating impurity was taken into account in the study of metabolic transformations using P45Oox, which is discussed below.

M-end sequence: -ATTATROI I SSOS5MREO-- (ZEO IU MO: 3)

Internal sequence: --MOVI MAO OVAAA- (ZEO IU MO: 4) 4.2. Allocation of MAORN-R.450-OKSMPorepUkKTazn

MAORN-P4i50-oxidoreductase is bound to OBAE-sepharose on a column of the ER/5-100-5erpagose brand and subjected to elution by adding buffer A with 0.5M KSI. The reductase is then purified to homogeneity on a 2',5'-AOR-Zerpagose 4B column (Rpagtasia), as previously described in the work of Naikieg apa MopPeg,

RiIapi Rpuzioi!. 96:10-17, 1990, and concentrated to about 15 units/ml. 4.3. Preparation of soluble uridine diphosphate glucose (SHORO) glucosyltransferase

Glucosyltransferase is partially purified by fractionation with ammonium sulfate of the supernatant after centrifugation obtained during the preparation of microsomes. The glucosyltransferase fraction is precipitated at 4095-6095 (MNa) at 250°C and dissolved in 5 ml of 50 mM tricin (pH 7.9), 2 mM OT and dialyzed against 2 liters of the same buffer for a day. 4.4. Restoration of cytochrome P4A50ox activity

Restoration of the enzymatic activity of microsomal cytochrome P450 is carried out by introducing cytochrome P4i50 and the corresponding MAORN cytochrome P.i50O-oxidoreductase into lipid micelles. A mixture of lipids can be used, but in the case of cytochrome P4B50ox dilauroylphosphatidylcholine (OIRPS) provides the best enzyme activity. The number of correctly formed cytochrome complexes

P4aB5boh and MAORN cytochrome P450 oxidoreductase is the rate-limiting factor. Excessive amounts of oxidoreductase and concentrated enzyme solutions are used to ensure a sufficient number of active complexes.

The functionally reduced enzyme is obtained using the following components:

Cytochrome 20μg/ml in dialysis buffer

P4ABoOoh:

MAORN 100 μg/ml in 5 MM potassium-cytochrome P450-phosphate buffer (pH 7.9) oxidoreductase from zogdapyt risoog

Lipid: 10mg/ml sonicated dilauroylphosphatidylcholine in 5OMM tricine (rin7,9)

MAORN: 25mg/ml in NgO 14b-oxime, 0O.01μCi/μl, 394mCi/mmol enzymatically obtained from (O-1423-I-tyrosine using reduced

5 μl of the lipid suspension prepared and purified by PCR is mixed in an Eppendorf vessel with 5 μl of MAORN cytochrome P450 oxidoreductase (0.075 units), 10 μl of cytochrome P45boh solution (approximately 0.4 pmol) and 0.5 μl of ""C-oxime (0.014 μCi/μl, 394 mCi/mmol). The final volume is adjusted to 30 ml using 50 mM tricin (pH 7.9) and the enzymatic reaction is induced by adding 1 ml of MAORN solution. Control samples are prepared by excluding

MAORN of cytochrome P.4i50-oxidoreductase or MAORN from the reaction mixture. The vessels are incubated with constant gentle shaking at 30"C for an hour. After incubation, the reaction mixtures are applied to thin-layer chromatography (TLC) plates covered with silica gel (Silica gel 60 Ez25l4, MegsK) and developed using a mixture of ethyl acetate/toluene (1:5 volume /volume) as the mobile phase. The plates are placed on phosphor (luminophore) screens and stored for a day, and the resulting products, p-hydroxyphenylacetonitrile and p-hydroxybenzaldehyde, are visualized using a ZTOKM 840 phosphor photoreceptor from MoiIesshag Bupatisv.

Next, cytochrome RibBOox, inserted into lipid micelles, catalyzes the conversion of -p-hydroxyphenylacetaldehydedoxime into p-hydroxymandelonitrile, which dissociates to p-hydroxybenzaldehyde and

NM. This shows that cytochrome P45boh is a multifunctional protein that catalyzes both the conversion of p-hydroxyphenylacetaldehydedoxime to p-hydroxyphenylacetonitrile and the conversion of p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. The activity of P4i5Oox completely depends on the presence of MAORN-P450 oxidoreductase and MAORN. Sodium dithionite (10 mM) does not contribute to the conversion of p-

hydroxyphenylacetaldoxime. If the enzyme is not dialyzed prior to reconstitution, this causes a relative increase in the accumulation of p-hydroxyphenylacetonitrile compared to p-hydroxybenzaldehyde. 4.5. Reproduction of the complete pathway of durrin synthesis from the starting amino acid tyrosine and migo

The final reaction mixtures contain: 3 µl of isolated, recombinant P450-tuv (bpmol, heterologously expressed in E. soya and isolated as described in NaiKieg et al., Agop. Biospet. Viorpuv. 322: 369-377, 1995), 100 µl of isolated and dialyzed P45boh (approximately 0.4 pmol), 5 μl. MAORN-P450-oxidoreductase (0.075 units), 1 μl of partially purified SHORO glucosyltransferase from sorghum, 5 μl of OROS (10 mg/ml in 50 mM Kry (pH 7)), 0.25 μl of (O-C|-tyrosine (0.05 μCi/mmol , 443mCi/mmol, Ategzpat), Zmkl SHORS (ZZmg/ml in 50MM

Kr, (pH7)) and ZmKl castanospermine (2mM in 50mM Kr, (pH7)) The components are mixed by resuspension, and if necessary, the final volume is adjusted to 30kL using 50mM Kr (pH7). The enzymatic reaction is started with 1 μl MAORN (25 mg/ml).

Durrin is also synthesized through the reduction of P450ox with p-hydroxyphenylacetaldehyde oxime (with the exception of

Ra4bBOtuv and tyrosine from the reaction mixtures, all additional components are left unchanged). These samples contain either 0.5 μl of HS-12C|-p-hydroxyphenylacetaldehydedoxime (0.014 μCi/μl, 394 mCi/mmol) or 3 μl of unlabeled p-hydroxyphenylacetaldehydedoxime (20 mM) as a substrate for P450ox. In the latter case, the radioactive label is μl (0-1221-(0ОРС) (0.025μCi/μl, 287mCi/mmol, Ategzpat). All reaction mixtures are prepared in duplicate. After incubation for 1 hour at 30"С, each set of reaction mixtures is applied on plates for thin-layer chromatography. The first set of reaction mixtures is analyzed using ethyl acetate/toluene as a solvent according to example 4.5. The second set of reaction mixtures is analyzed using a solvent system consisting of ethyl acetate/acetone/dichloromethane/methanol/water (20/15/6 /5/4, all - by volume) to achieve separation of the hydrophilic product of durin from tyrosine and from hydrophobic intermediates.

Radiolabeled substrates and products are visualized using a Z5TOEM 840 phosphor photoreceptor.

The mixed use of isolated PA50 and P450 in reduction experiments using radiolabeled tyrosine as a substrate results in the production of p-hydroxyphenylacetonitrile and p-hydroxybenzaldehyde. This shows that R.i5O-tuv and Ra4Bboh are able to act together ip mygo. P-hydroxyphenylacetaldoxime, formed by P4A50-tuv, is thus effectively used as a substrate for

R4BOoh. In the absence of MAORN-P450 oxidoreductase or in the absence of MAORN, no activity was observed.

The production of durrin using p-hydroxyphenylacetaldoxime as a base was carried out by reducing P450ox together with partially purified soluble OOPO-glucosyltransferase in the presence of MAORN and OOPO. A cDNA clone from sorghum encoding a SHORO-glucosyltransferase that specifically uses p-hydroxymandelonitrile as a substrate is missing. Accordingly, in this study, a crude extract of soluble SHOROS-glucosyltransferase from sorghum was used for glucosylation of p-hydroxylmandelonitrile and to demonstrate the restoration of the complete pathway of durin biosynthesis.

The applied radiolabeled p-hydroxyphenylacetaldoxime was completely metabolized. Castanospermine was added to inhibit glucosidase activity, which is observed during the preparation of POORO-glucosyltransferase. In addition to the thin-layer chromatography system used earlier to separate hydrophobic compounds, an additional thin-layer chromatography system is used to separate hydrophilic compounds such as durin. The p-hydroxymandelonitrile formed during the reduction was partially converted to durrin, as evidenced by the formation of a radiolabeled compound that comigrates with true durrin. The identification of this radiolabeled compound as durin was confirmed by its decay in the absence of castanospermine and the formation of a comigrating radiolabeled product when the experiment was repeated with radiolabeled OROS instead of radiolabeled p-hydroxyphenylacetaldoxime. Radiolabeled OORO nonspecifically contained a number of relatively hydrophilic compounds. Due to the instability of p-hydroxymandelonitrile, its conversion to durin was experimentally detected due to the disappearance of p-hydroxybenzaldehyde.

When radiolabeled p-hydroxyphenylacetaldoxime was used as a substrate in addition to durrin, many unrecognized hydrophobic radiolabeled compounds were formed. The formation of these compounds occurs in the absence of SHORPO, but requires the presence of a soluble extract, which shows that the SHORPO-glucosyltransferase extract has additional enzymatic activity. Glucosylation of the phenolic group of p-hydroxymandelonitrile leads to the formation of p-glucopyranosyloxymandelonitrile. No radiolabeled product was observed comigrating with the authentic p-glucopyranosyloxymandelonitrile standard. Glucosidase activity, which has POROB glucosyltransferase extract, is effectively inhibited by castanospermin.

After the IP was restored, the conversion rate for the P450-tuv (SUR79) is 230 min"! (5irrezep ei ai,

U. Vioi. Spent 270: 3506-3511, 1995). The partial transformation of P450oh into its denatured P420 form prevents the determination of this indicator. With the use of the microsomal system, the values of Kt and Mmax FOR the production of p-hydroxymandelonitrile from tyrosine, p-hydroxyphenylacetaldoxime /-( and p-hydroxyphenylacetonitrile are 0.03, 0.05 and 0.10 mM and 145, 400 and 5Onmol mg of protein "h", respectively (Meieg ei ai, U. Visi. Spet. 254: 8575-8583, 1979).

The complete pathway of durrin biosynthesis, starting from the original amino acid tyrosine, was reconstructed by combining Rai5O-tuv, P4BbB0ox, MAORN-P450 oxidoreductase in OGRS micelles with OORO-glucosyltransferase, tyrosine, MAORN, SHORO and castanospermine. Tyrosine is converted to p-hydroxyphenylacetaldoxime using RagbBO-tuv, which is further converted to p-hydroxyphenylacetonitrile and p-hydroxybenzaldehyde using P45box. A certain amount of p-hydroxyphenylacetonitrile accumulates, while all of the p-hydroxymandelonitrile formed is converted to durin and some unidentified compounds. In this series of experiments, the stoichiometric ratio between РА50-tuv and РА5Ох is approximately 15. Therefore, there is nothing surprising in detecting the accumulation of -p-hydroxyphenylacetaldoxime in the reduction experiment. The observed accumulation of p-hydroxyphenylacetonitrile is unexpected because previous experiments with sorghum microsomes showed that p-hydroxyphenylacetonitrile is difficult to accumulate and separate. Partial denaturation or inactivation of isolated P4b5Oox may explain why p-hydroxyphenylacetonitrile accumulates in reduction experiments with isolated P4A5boh. 4.6. Substrate binding

The identification of RaBbOx as a multifunctional enzyme that converts p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile with p-hydroxyphenylacetonitrile as an intermediate prompted investigation of the ability of RaibBbOx to bind the substrate. For p-hydroxyphenylacetaldoxime, a type I reverse spectrum was obtained with a minimum absorption at 381 nm and a maximum absorption at 418 nm, which indicates a change in spin from high to low after adding the substrate. During incubation, the amplitude increased and reached a stable maximum after approximately 45 min.

When p-hydroxyphenylacetonitrile was added, no substrate binding spectrum was obtained.

P4BbOox turned out to be significantly more labile compared to other RA50 enzymes isolated from sorghum.

The isolated RaiBbOoh gives the reverse spectrum of substrate binding! type when incubated with p-hydroxyphenylacetaldoxime. The extinction coefficient Ea2o-zzo, which corresponds to a complete transition from one spin state to another, is 130 mm cm cm!. In the obtained spectra of substrate binding, the maximum amplitudes are approximately twice as large as theoretically calculated, even if we assume a complete change of high spin to low spin. This discrepancy shows that the P450ox concentration was underestimated when determined by the 450nm peak in the carbon monoxide binding spectrum. Or RA20 form

P4Bboh is able to bind the oxime and, thus, contributes to the increase of the formed binding spectrum of the substrate. The latter possibility may explain why maximal amplitudes can be obtained only after prolonged incubation.

Previously, it was reported about R.i.50-mediated dehydration of aldoximes to nitriles using liver microsomes (OeMazieg eyi ai, U. Ogd. Spet. 5074-5075, 1992). The main difference between the liver microsome system and P450ox is that the former requires strict anaerobic conditions, while the latter requires aerobic conditions, catalyzes the subsequent C-hydroxylation reaction and metabolizes the (e)- as well as the (2)-isomer. Under anaerobic conditions, liver microsomes give a weak spectrum of type I.

When MAORN or dithionite is added, a distinct Bohey peak at 442-444 nm is formed. From this we can conclude that it represents the main active varieties of P450 in the Re (II) state. Examination of the spectrum of P45Oox under anaerobic conditions does not reveal the formation of a 442 nm absorbing complex, but the presence of MAORN is necessary for catalytic activity, which shows that P450ox must also be in the Re(II) state in order to mediate the dehydration reaction.

Example 5: Formation of a cytochrome P450 A-type probe

Polymerase chain reaction (PCR) was performed on plasmid DNA isolated from a unidirectional library of plasmid cDNA (Ipmigodep) from etiolated seedlings of Zohapit bBisoiotsiI) Moepsip 1-2 cm tall using largely degenerate inosine (I) containing primers that selectively select cytochromes Ra .50 A-type (Meizop apa Oytg5i, Ogyd Meiaroijist apa Ogyd Ipiegasiop5 12: 189-206 (1995)).

Primer 1 (sense strand) with the sequence 5-YaaS(aaAAATTSOTTMPSSMAdAMamtt-3 (5EO IU MO:5) covers the agreed amino acid sequence EHREKE (ZEO IO MO:6), in which X is any amino acid.

Primer 2 (antisense strand) with the sequence 5'YASOaaZATSSPASAPMSKMOSKMOS-3 (5EO 1O MO:7) covers the agreed amino acid sequence OKEHOHO (5EO IYUO MO:8). Primer 1 and primer 2 had EsokKiI and Vatni sites at the ends, respectively, which ensured cloning of only polymerase chain reaction (PCR) products formed from both primers in fermented EsovI/Vatni rVishevzsgiri II ZK (5igagedepe).

Polymerase chain reaction (PCR) was carried out in two consecutive stages. Step 1 using primer 1 and the standard T7 primer 5-AATACEASTSASTATAS-3' (5EO IO MO:9) enriches the cDNA pool encoding cytochromes P450 A-type. Step 2, including primer 1 and primer 2, formed mainly one chain of approximately 100 bp specific for cytochromes P450 A-type. The polymerase chain reaction for stage 1 was carried out in a total volume of 100 μl, which contained 595 DMSO, 200 μM deoxynucleoside triphosphate (aMTP), 200 pmol of primer 1, 100 pmol of standard T7 primer, 2.5 units of Tad DNA polymerase in PCE buffer and 1 μl of 100-fold dilution plasmid DNA from the library

KDNK. RSE for stage 2 was carried out in a total volume of 100 μl, which contained 596 DMSO, 200 μM amtR, 200 pmol of primer 1 and primer 2, 2.5 units of Tad DNA polymerase in RSK and 1 μl of the product obtained as a result of RSE. stage 1. During both stages of the ROK in one cycle of 5 min. at 957C, 35 cycles followed

Zosek at 95"C, 1 min. at 50"C and Zbosec. at 72"C. Approximately 100 bp of the RSE product of stage 2 was excised from a 2905 agarose gel and re-amplified before cloning in rVishpezsgiri. Of the 19 sequenced clones, 10 had very high sequence identity at the amino acid level with cinnamic acid hydroxylase (SUR 74) and therefore were not subjected to further study. Comparison of the remaining 9 sequences divided them into two groups of 8 and 1 sequences, which were designated "12" and "7", respectively. The gene-specific primer of the sequence "12", located between primer 1 and primer 2: 5 -4SssaSpATSSSASTASTASYASTOOO- 3 (ZEO IU MO:10), and primer 5'-YASOSATSSTTTTTTTTTTTTTTTTM-3' (ZEO IU MO:11), which have at the ends

Vatni, used for amplification "12" gene-specific fragment from approximately 500 bp. RSC-stage 1 was cloned in rVinezsghir. In a similar way, a gene-specific fragment for "7" was obtained using the "7" gene-specific primer 5-ЯСасАТССССАСААДОСасСАас(а-3 (5EO IU MO 12) and the primer 5-ЯСасАТССТTTTTTTTTTTTTTU-3 (5ЕБО IO MO:11).Inserts were labeled, Oidohidepip-11-4O0TR (Voephipdeg Mapppeit) by PCR amplification with standard 17 and TZ primers according to the manufacturer's instructions and used to screen cDNA libraries.

Example 6: Library screening and DNA sequencing

All filter hybridizations were performed using the OIS system (Voepgipdeg Mapppeit). Layers of colonies were prepared using a nylon membrane (Voepgipdeg Mappeit) and hybridized for a day at 68"C in 5x55C (sodium chloride and citrate solution), 0.190 M-lauroylsarcosine, 0.02905 sodium dodecyl sulfate (505). 195 ViosKipd Keadepi (Voepgipdeg Mappeit) Filters were washed twice for 15 minutes in 0.1x55C, 0.190 505 at 65"C before detection. Full-length clones for "12" and "7" were obtained, as evidenced by sequence analysis. Sequencing was carried out using a labeled Tpepto bBedcepase Ripogasepi primer cycle sequencing kit (7-deal astrP) (Ateghzpat) and analyzed for AG P-Ehrge55 (Rpagtasia). Computer analysis of the sequence was carried out using the program in OSO UMizsop5zip zedcepse Apaiuzi5 RasKkade. full-length cDNA sequence

P4bBOoch and the derived amino acid sequence of the coding region, obtained as a result of sequencing of nucleotide "12", are presented in ZEO IO MO: 1 and 5EO IO MO2, respectively.

Example 7: Expression in E. Soya

Expression vector reP19910! (Vagpe5, Meipod5 ip Epgutoiodu 272: 3-14, 1996) obtained from Dr. Henry

Barnza (Og. Nepgu Vagpe5 (Zupipebis sSepeiis5/Ittipe Sotrieh Ipsogrogayop. Zap Oiedo, SA)). This plasmid contains the Ias7 promoter fused to the short leader sequence of gene 10 of 17 bacteriophage, 9101, which has been recorded as an excellent leader sequence for the expression of various heterologous proteins (Oiip et al., 1988). "7" and "12" were changed by RSE using Ruho polymerase (Voepgpeg Mappeit) to insert a MaeI site in the start codon and to change the stop codon to an osgE stop codon immediately followed by a Nipai site. To create an expression clone for "7" we used primer C (sense strand) 5-СаСО)ааААТССАТАВАСОаSATSATTAST ССТОТОСОТСОSasto-3 (5EO IU MO:13) and primer 4 (antisense strand) 5'-СаСААаСТТАТТАСТСТСААСОаасвАСССТ-3 (5EO IB'MO:14 ).

Primer Z introduces silent mutations in codons 3, 4, and 5 to reduce the 5/C content around the translation initiation site and the Watni site just before the Mah! site The resulting RSOK fragment was fermented

Vatni and NipaPy and ligated into fermented Vatni and NipaPy! rVisezspri and controlled by sequencing to exclude RSE errors. Similarly, "12" was introduced into the rVisezspry using primer 5 (sense chain) 5-СаСацпАТССАТАССААССААСаСААСССССАСАстосто-3' (5EO IU

MO:15) and primer 6 (antisense strand) 5-СаСААаСТТАТАТАТОСОСООСОС vat сСстТатАТТТОс-3 (5BEO IЮО MO:16). Primer 5 introduces silent mutations in codons 2, 3, 4 tab, and primer b introduces silent mutations in the last 2 codons to reduce the content of 5/C. Inserts were cut using mae! and NipaI and ligated into fermented Maei and Nipap! reP1994910. Expression plasmids were transformed into E. soy UM109 cells. Single colonies were grown for a day in II B medium containing 100 mg of ampicillin/ml at 37"C and 5 ml of yesterday's culture used for inoculation X00 ml

TV medium containing 50 mg/ml ampicillin, 1 mM thiamine, 1 mM isopropyl-pD-thiogalactopyranoside and mM 5-aminolevulinic acid. Cells were grown at 287C for 48 hours at 125 rpm. Iml. E. soybeans were transformed using expression sequences "7"; "127 and rBZRI9d10Y were precipitated by centrifugation (2000 g, 10 min), washed and concentrated 10 times in 5X0 mM tricine pH 7.9 and incubated with 14 pKi of 0-19 p-hydroxyphenylacetaldehydedoxime with a specific activity of 394 mKi/mmol at 30 for 30 h. The incubated mixtures were extracted with ethyl acetate , applied to a plate for thin-layer chromatography (Silica gel 60 Rov4., MegsK), developed using a mixture of ethyl acetate/toluene (1:5 v/v) as a mobile phase and visualized using a 5TOEM 840 apparatus from MoiIesshag bupatisv.

E. soy transformed with the sequence expressing "12" was able to convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyphenylacetonitrile.

CO difference spectra of solubilized E. soybean spheroblasts expressing P450ox had the highest peak at 417 nm and the lowest peak at 457 nm. In general, the CO-spectrum with a peak of optical density around 420 nm is an indicator of cytochrome P450 in a non-functional conformation (Itai ei ai, Eig. U. Viospet. 1: 419-426, 1964). The presence of the highest peak at 417 nm indicates the presence of most of the expressed cytochrome P450 in a non-functional conformation. The apparent shift of the optical density peak from 450 nm to 457 nm may be due to the presence of a large amount of cytochrome Pi450 in a non-conformational state. Based on the peak at 457 nm, the output of 5 Onmol P45Ooch per 1 liter of E. soybean culture in 65 hours was calculated. 5 μl of membranes isolated as described in the work of NaiKieg ei ai, Agspime5 oi Viospetivzigu apa Viorpuzisv 322: 369-377, 1995, from E. soii expressing "12", was reconstituted with 0.225 units of MAORN-cytochrome P450-reductase, 50 μg of MAORN . After incubation at 30"C for 30 minutes, the reaction mixture was applied to a plate for thin-layer chromatography and analyzed as described above.

Regeneration of the membrane from E. soya expressing "12" led to the accumulation of p-hydroxybenzaldehyde, which is a stable dissociation product of p-hydroxymandelonitrile, the last intermediate in the biosynthesis of the cyanogenic glucoside durin. This indicates that "12" is cytochrome P4i50, which catalyzes the conversion of p-hydroxyphenylacetaldehydedoxime into p-hydroxymandelonitrile. cDNA is denoted as

Ra4Bboh. The clone, which includes the described cDNA P45BOH, was deposited on January 10, 1997. to the German Collection of Microorganisms and Cell Cultures (05M2-Yuetsivspe Zattiypd mop Mikgoogdapiztep ipa 7eikiyshShgep sSitrN, Mazspegodeg UUyed 16, 0-38124 Vgapp5zspmeid) under inventory number О5M 11367.

When radiolabeled p-hydroxyphenylacetaldoxime was injected into P45Oox-transformed E. soybean cells, p-hydroxyphenylacetonitrile accumulated in E. soybean cells expressing P45Oox. E. soybeans, which do not contain endogenous cytochromes P4-50 or MAORN-cytochrome P4i50-reductase, showed support for the catalytic activity of heterologously expressed cytochromes P4i50. Two soluble flavoproteins BE. Soybeans, flavodoxin and MAORN-flavodoxin reductase transfer reducing equivalents to recombinant cytochromes P4 and 50. Reconstitution of isolated E. soybean membranes or purified recombinant enzyme with sorghum MAORN-cytochrome P450 reductase leads to the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile, whereas cells of EE. soybeans expressing P4i5Boh metabolize p-hydroxyphenylacetaldoxime only to p-hydroxyphenylacetonitrile. The inability of E. soy to support the conversion of p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile is the first example that the BE system. soybean flavodoxin/MAYURN-flavodoxin reductase, unable to support the catalytic activity of microsomal cytochrome P450 reaction. Previous reconstitution experiments with membrane and soluble fractions of E. soya cells expressing P4i50ox showed that the soluble flavodoxin/MA ORN-flavodoxin reductase system only supports RaBOox in the conversion of p-hydroxyphenylacetaldoxime to p-hydroxyphenylacetonitrile and that an inhibitory factor that prevents further reaction hydroxylation, absent in the soluble fraction. The inability of E. soya to maintain full P450ox activity may reflect the atypical catalytic reactivity of P4A45Oox. In BE cells. soybeans transformed with P4iB5box or the expression vector accumulates a compound with slightly lower mobility than p-hydroxyphenylacetaldoxime. This shows that E. soii can metabolize p-hydroxyphenylacetaldoxime independently of the presence of PA5Oox.

Example 8: Purification of recombinant P4A5boh (SUR7TE1)

Spheroblasts from 2 I E. soybeans expressing P45boh were subjected to phase separation under the influence of temperature using 195 Triton X-114 as described above (NaiKiegei ai, Agspimez5 oi Viospe pizigu apa Viorpuvzisv

Z22: 369-377, 1995). The upper phase containing P45Oox was diluted 100 times in 10 mM KR, pH 7.0, 0.059 reduced Triton X-100, 1 mM OTT, 0.5 mM phenylmethylsulfonyl fluoride (PM5BE), the pH was adjusted to 7.0 with acetic acid and added to 1 bml fast-flow column SM-Zerpagose (Rpaptasia), equilibrated in buffer O (10 mM KR, pH 7.0, 0.295 triton X-114, 0.0595 reduced triton X-100, 1095 glycerol, 1 mM

OTT, 0.595 RM5B). The column was washed in buffer O, and P45box was eluted with a linear gradient of 0-1 M KSI (3500ml) in buffer O. Combined fractions containing P45box (43ml) were used for recovery experiments, and electroeluted recombinant P450ox was used to produce antibodies in chickens.

Purified recombinant P450box, reduced by MAYURN-cytochrome P450-reductase in OI RS catalyzes the conversion of p-hydroxyphenylacetaldehydedoxime to p-hydroxymandelonitrile in the presence of MAORN, as described in example 4, section 4.2 for the purification of plant enzymes.

Example 9: Durrin expression in transgenic plants Agabridorvi5 and tobacco 9.1 Construction of vector plasmids

Three binary vectors for Adgobasiegiit Shstetiasiep5-mediated transformation were generated, namely - PPAR111.79, pPAR221.71ЕЕ1 and pПАР111.79Е1.

For the construction of pPAP111.79 cDNA clone from P45Otuk (M/095/16041; Kosi ei ai, Agsp Viosnet Viorpuz 323:177-186, 1995) is first excised with the help of EsokIi and the pKT101 site is introduced into EsokIi (Korgeg ei ai, Mysieis Asiaz

Kezeagsp 15:5890, 1987) for the functional attachment of cDNA to the 355-promoter and plasmid pKT101,79, which generates the Samu polyadenylation signal. Before the introduction of P45Otuv cDNA, part of the reET101 polylinker is removed by fermentation with the help of Zai and Khbrai, after which the "blunt" ends obtained by the Klenov method are religated, thus leaving only EsoK!I and Xpo!I sites. P4A5BoOtuv, including the 355-promoter and Samu polyadenylation signal, was excised from pET101.79 using 5rp1.

To create plasmid pPAP111,79, the ends are "blunted" with Klenov polymerases, and the resulting fragment is ligated into the EsokI cut plasmid pPAP111 (Na/dikiveuis: ei ai, Riapi Moi Vio! 25: 989-994, 1994), the ends of which were also "blunted" Klenova polymerase and dephosphorylated.

To construct pPAP221.71 E1 cDNA clone P4i5boh is first excised with the help of Krpita Hbvai and ligated in Krp and Hrai sites of reET101, forming pET101.71 E1. P450ox, including the 355 promoter and Samu polyadenylation signal, is then excised from pKT101.71 E1 with Nipap and ligated into Nipap | site of pPAER221 (Naddikiemisya ei ai, Riapi Mo! Vioi 25: 989-994, 1994), thus forming pPAER221.71 E1.

To create pPAPRI11.79.71E1, a clone of the cDNA RaBbbBbOox, including the 355-promoter and the Samu polyadenylation signal, is excised from pPAP221.71E1 using Nipai, "blunted" with polymerase

Klenova and ligated in Zztai site pPAR111,79. 9.2 Agaridorviz transformation (PpaIapa

The binary vectors pPAP111, pPAP221, pPAP-111.79, pPAP221.71E1 and pPAP111.79.71E1 are introduced into the strain

Adhorasiecht isheitasiep5 C58C1/rou3850 by electroporation, as described in the work of UUepishp apa Eogae,

Journal of Asian Studies 17: 8385, 1989. Agaridorvi hyalap, ecotype of Soiotbria, is transformed by vacuum infiltration, practically as described in the work of Vesptoiai ei ai, Moiieshag Vioiodu apa Sepeis5 316:1194-1199, 1993. Transformants are selected on plates for mass- spectrometry containing 50 μg/ml kanamycin for pPAP111 vectors, or 200 μg/ml gentamicin sulfate for pP2P221 vectors.

4-6 weeks after germination, plants resistant to kanamycin or gentamicin are transplanted into the soil. 9.3 Transformation of Misoiap iarasit suHNapii

Misoiyapa yarasit sm Khapiyas are converted almost according to the method of conversion of sheet discs described in the work of Zmar ei aI, Meijoaz ip Riapi MoIesshag Viiodu, Soii Zrgipd Nagrog, pp. 55-60, 1995, using Adgobasiect steiasiep5 C58C1/rM3850 converted with the help of rRagRI11. 79, pPAP221.71E1 or pP2P111.79.71 E1. 100 μg/ml of kanamycin is used for the selection of transformants using the pPARI111.79-vector, 100 μg/ml of gentamicin sulfate - for the selection of transformants using the pPaP221.71E1 vector, and 5 Ωg/ml (0-418 - for the selection of transformants using the pPARP111.79.71 E1-VrVctor3. After the formation of the root system, the plants are transferred to the soil and grown in a greenhouse. 9.4 Definition of durin

Durrin content is determined using spectrophotometric cyanide analysis, previously described in the work of NaiKieg ei ai, Riapi Rpuzio! 90: 1552-1559, 1989, only in this case 5-10 mg of leaf tissue is frozen and thawed three times before adding 0.1 mg of r-d-glucosidase, Type I! (Bidta). 9.5 Analysis of transgenic plant Agaridorviv5 Ttaiyapa

Detached leaves of A. Ipaiyapa are impregnated with 2 μl of (11-14C)-tyrosine (0.05 μCi/μl, 443 mCi/mmol, Ateg5pat) and left for a day in 100 μl of H2HO in closed Eppendorf vessels. Metabolites are extracted by boiling 9095 methanol for 5 min, the extracts are concentrated and applied to plates for thin-layer chromatography (Silica gel 60 254). Metabolites are separated in three different solvent systems, depending on their hydrophobicity/hydrophilicity. The solvent system ethyl acetate/acetone/dichloromethane/methanol/water (20/15/6/5/4, all by volume) is optimally used to separate various cyanogenic glucosides. The solvent system isopropanol/ethyl acetate/water (7/1/2) separates various glucosinolates. The solvent system toluene/ethyl acetate (5/1) separates hydrophobic intermediates. Radiolabeled substrates and products are visualized using a ZTOKM 840 phosphor photoreceptor (MoiIesshag Bupatisv, OA).

Methanolic extracts of leaves of A. Tpaiiapa, which were analyzed by thin-layer chromatography of plants transformed with the help of Pa4bBOts and P4bBOx with the use of A. istheiasipe5

C58C1/roUZ850/rRYARI111.79.71E1, reveal the presence of a new compound that comigrates with the cyanogenic glucoside durin. Treatment of detached leaves with radiolabeled tyrosine shows that the radiolabel is contained in a band that comigrates with durrin. Colorimetric cyanide analysis of the whole leaf tissue of the transformed plant (Gatregi ei ai, 1975, Apaimis Spetivzigu 47, 917-919) reveals that T1 transformants contained from 1 to bnmol cyanide/mg raw mass. In contrast to them, the control plants transformed only with the use of A. Shpteiasiap5 C58C1/roUz3850/rPAP1I111 revealed a cyanide content of 1.2250.35 nmol per mg of raw mass. Cyanogenic glucosides were not detected in wild-type plants of A. ippaiiapa, and the apparent level of cyanide found in control plants most likely reflects the presence of thiocyanates.

Thiocyanates are breakdown products of glucosinolates and are known to give a false positive reaction in the colorimetric cyanide assay (Hervey, 1974, Appl. Science 19, 272-274).

Production of a large amount of cyanogenic glucoside durrin as a result of the introduction of P45Otuv et

RabBOoch, which was observed on the example of A. Tpaiiap, testifies to the presence of PSHOR-glucosyltransferase, which glucosylates p-hydroxymandelonitrile formed in the correct position. Since the stereospecificity of the glycosyltransferase is not known, it is possible that the cyanogenic glucoside formed is actually taxiphylline, which is an epimer (mirror isomer) of durin.

When P45Otuv is injected with C58C1/ruZ3850/pP2P111.79 A. stegasipsis into A. Taijapa, a compound that comigrates with a tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate, accumulates in large quantities. This indicates that the introduction of P4iB5Otuv leads to the formation of p-hydroxyphenylacetaldoxime from tyrosine. In this case, the tyrosine-derived oxime is further metabolized by enzymes via glucositpate to p-hydroxybenzylglucosinolate. This clearly shows that the oxime enzyme in the glucosinolate pathway has low substrate specificity with respect to the structure of the side chains and that the glucosinolate profile is generally determined by the substrate specificity of the first cytochrome P450 in this pathway.

The substrate specificity of P450oh is not as narrow as that of P45Otuv. P450ox can metabolize other amino acid-derived oximes, for example, the phenylalanine-derived oxime, phenylacetaldoxime, while

RAaBOtuvu can only metabolize tyrosine. When P450ox is introduced into glucosinolate-producing plants, it can thus be expected that cyanogenic glucosides accumulate as oximes formed from amino acids in the pathway of glucosinolate biosynthesis.

Since P45Otuv and P450ox interact with enzymes and precursors in the glucosinolate biosynthesis pathway, it is expected that the glucosinolate profile should also be altered. 9.6 Analysis of the transgenic plant Misoiapa iarasit cm Khpapii

Microsomes isolated from tobacco plants, which were transformed with pRa2P111.79 and functionally express SUR79U, catalyze the formation of tyrosine in p-hydroxyphenylacetaldoxime. Methanol extracts from detached tobacco leaves expressing SUR79 and treated with radiolabeled tyrosine contained three additional labeled bands compared to wild-type tobacco plants, as demonstrated by thin-layer chromatography analysis using the isopropanol/ethyl acetate/water (7/1/2) solvent system.

An additional three bands comigrate in thin layer chromatography with labeled bands detected in wild tobacco treated with radiolabeled p-hydroxyphenylacetaldoxime. When analyzed in the solvent system toluene/ethyl acetate (5/1), it is confirmed that one of the bands is p-hydroxyphenylacetaldoxime, as evidenced by comigration with cold standards. The identity of the two additional bands has not yet been revealed, but judging by their mobility in both solvent systems, they should be less hydrophobic than p-hydroxyphenuiacetaldoxime and most likely are glycosylated derivatives of p-hydroxyphenylacetaldoxime. Analysis of SMP7ZU-plants confirms that SMP7Z can be functionally expressed in tobacco and that a certain amount of free p-hydroxyphenylacetaldoxime is formed, but most of the formed p-hydroxyphenylacetaldoxime is metabolized by plants.

Plants expressing P45Otuv and P450ox can be obtained by crossing plants expressing РА5Otuv with plants expressing P45boh. Or plants expressing RA5Otuv or

P4a4Bbooh, can be re-transformed with the help of A. iistetasieep5 C58C1/roM3850/pPAP221.71E1 or

A. Titegasiepe C58C1/reM3850/pPARI111.79, taking advantage of the fact that two cytochrome P4A50 sequences bind to two different non-exclusive resistance markers, priiI and assi.

Example 10: Durrin expression in transgenic maize 10.1 Creation of vector plasmids

The next two vectors, pSIV 9842 and pSIV 9833, are produced during the biolytic transformation of corn using P45Otuv and P4A5Oox. pSIV 9842: cDNA clone encoding P45Otuv, cloned in EsokK! the site of rVicezsgiri II ZK, as described in

UUO095/16041, are used for the formation of the Vat site in the start codon of the ATO codon and the Ba II site in the stop codon by RSE. Watni-Wa! Its fragment, containing the P45Otuv gene, is cloned in Vatna and Va II break of pSIV 9805, a plant expression vector based on pOS19, engineered with the help of ATSHI/Mo/Ausi sites 256 pairs of nucleotides before Nipaia! site and through 778 p.n. after Water of a site containing a metallothionein-like promoter described in EP-A-452269 and a 355 terminator. рСИВ 9833: P450х кКДНК clone according to the Example, cloned in a Moi-V5iIHI site of rsOMAI! (Ipiygodep) is used to form the Vat site in the ATO start codon and the Vadi II site in the stop codon. Vatni-Va II fragment containing the P45boh gene was also cloned into pSIV 9805 trimmed with the help of Vatni and Va. 10.2 Methods of corn transformation

Embryogenic callus cultures type (Sgeep ei ai, 1983; UMap ei ai, 1994) Lancaster inbreeding was initiated from underdeveloped embryos, 1.5-2.5 mm long. Embryos are aseptically excised from the sterile surface of greenhouse-grown spikelets approximately 14 days after pollination, placed in Duncan's callus initiation medium with 295 sucrose and 5 mg/L chloramben and cultured in the dark.

The embryogenic reaction is removed from the explants after about 14 days and placed on Duncan's stabilizing medium with 295 sucrose and 0.5mg/l 2.4-4. After 4-8 weeks of weekly transplantation to update the stabilizing medium, high-quality compact embryogenic cultures appear. Actively growing embryogenic callus fragments are selected as target tissues for gene introduction. Fragments of callus are placed on target plates containing a stabilizing medium of 1295 sucrose approximately 4 hours before the introduction of genes. Callus fragments are placed in circles, 8 and 10 mm from the center of the target tablet. Plasmid DNA is deposited on gold microcarriers as described in the manual of BiRopi Wioiwiis5. Two to three μg of each plasmid is used in each b-dose preparation. Genes are injected into target tissue cells using the ROBZ-I000ONE Vioiistis5 device. The parameters of the Vioiisis5 device are as follows:

3mm between the rupture disk and the macro carrier, 10 mm between the macro carrier and the retarding screen, and 7 cm between the retarding screen and the target. Each target tablet is dosed twice with the use of 6b5 Orzi rupture discs. A 200x200 stainless steel sieve (MeMazieg-Sait, Mezh Vgypzm/isk, MU) is placed between the retarding screen and the target fabric. Seven days after the introduction of genes, fragments of target tissues are transferred from a highly osmotic medium to a selective medium containing 100-120mg/l ammonium glufosinate (Vavzia). All amino acids are removed from the selective medium. After 5-8 weeks on high-level selective medium, all developing colonies are transferred to medium containing 20 mg/L Vazia. Embryogenic callus is transferred every 2 weeks for 4-8 weeks, and then transferred to modified M5b medium containing 395 sucrose, 0.25mg/l ancymidol, 0.5mg/l kinetin without a selective agent and placed under light. Antsimidol and kinetin are removed after 2 weeks. Regenerating shoots, with or without roots, are transferred to Magenta boxes containing M5 medium with 3905 sucrose, and young plants with roots are eventually regenerated and transferred to soil in the greenhouse.

Example 11: Identification of P4A5Otuv homologues in glucosinolate-containing species by RSC

On the basis of the computer sequence determination of the P4A50 homologue of Agaridorziz E5T (inventory number T42902) and the P45Otuk homologue from zipari5, two degenerate oligonucleotide primers are designed, which allow amplification of fragments of RSC homologues of P45Otuv from genomic DNA species containing glucosinolate. The sense strand primer (5-saCOsAATTSAANSCISAAMYISAMUT-3) covers the conserved amino acid sequence of CRACKNI (5EO IO MO: 18) and includes the Esoki cloning site.

Antisense strand primer 2 (5-2COaSATSSASAISSISCUTTISSMOT-3) spans the conserved amino acid sequence TOKKOS (5EO IO MO: 19) and includes the Watney cloning site. PCR is performed on genomic DNA prepared using the Mysieop Rpuiorige Riapi OMA Ehigasiop kit with

Amsterdam. RSE is carried out in a total volume of 100 μl, containing 5906 DMSO, 200 μM amMtR, 200 pmol of each primer, 2.5 units of Tad polymerase in a buffer for ROK (50 mM KSI, 10 mM Ttgi5-HCII (pH 8.8), 1.5 mM

Mass" and 0.1 95 Triton X-100) using 1 μg of genomic DNA from pairs of Aibra, A. Ipaiapa, Vgazzisa pair",

Tgoraeoiit ta/)iz or M. tarasit cm Khpapii. RSE is carried out in four consecutive stages: stage 1: one cycle of 5 minutes. at 957C; stage 2: 5 cycles by Zosec. at 957C, ZOsec. at 557C, ZOsec. at 7270; stage 3: 30 cycles for ZOsec. at 957C, ZOsec. at 60"C, 3 sec. at 72"C; and stage 4: one cycle of 5 min. at 7276.

For the formation of a sufficient number of bands of approximately 100 bp. with T. and/)c5, stage 2 can be changed to cycles of ZOsec. at 957C, 30sec. at 50"C, ZoOsek. at 71"C. The RSK products are purified using the RIDDNISK ROK Rigiisayiop Kit (Oiadep), restriction digested with Esoka and Zatni, and separated on a 395 TAE agarose gel. Cut out the dominant chain at about 100 bp. and ligated into linearized with the help of EsokUVatni rVincezosgiri II ZK (5igaiadepe). Approximately 10 clones from each of the 5 samples are sequenced using a sequencing kit with a fluorescently labeled primer

TNepto Zedyepse RiIpogezsepi-IabePei Riiteg (7-dealga STR) (Ategzpat) and analyzed for AGR-Ehrgev5 (Rpaptasia).

Of the four glucosinolate-containing species, 5. aIra, A. (Shtayapa, V. Mari5 and T. ta|y5 identify RCE fragments encoding a conserved amino acid sequence,

CREAVN(I/R)MESZEMTI THEME OF ARIBEUTOACNOGS (BO 10 MO: 20 and 21, respectively) This aligned amino acid sequence is identical to previously identified homologous RA5Otuv sequences from 5. aiira and A. Tpaiiapa and is very similar to the amino acid sequence of RA5OIIUug. From a plant that does not contain glucosinolate, M. tarasit cm Khpapii, the RSC fragment encoding this agreed sequence was not identified. The presence of this homologous P45Otuv consistent amino acid sequence in the four glucosinolate-containing plant species indicated indicates that cytochrome P4i50 amino acid M-hydroxylase from the P45Otuv family converts parent amino acids or parent amino acids with extended chains to the corresponding oximes in glucosinolates. Formation of ROK fragments specific for homologues

P4iBoOtuv allows isolation of homologous cDNA or genomic clones from the appropriate libraries.

While this invention has been described in some detail by way of illustration and example for the purpose of better understanding, it will be understood that certain changes are possible therein without departing from the spirit of the formula.

LIST OF SEQUENCES

(1) GENERAL INFORMATION: () SUPPLIER: (A) NAME: SIVA-SE|SU AS (B) STREET: Kiureskeig. 141 (B) CITY: Basel (D) COUNTRY: Switzerland (E) POSTAL CODE (2IR): 4002 (E) PHONE: 41 61 69 11 11 (F) FAX: - 41 61 696 79 76 (3) TELEX: 962 991 (A) TITLE: Kouai Meiegipagu 5 Adgisinkiga! Opimegesiu (Royal University of Veterinary and Agricultural Sciences) (B) STREET: 40, TPpogmaiazepzme) (C) CITY: Frederiksberg (G) DISTRICT: Copenhagen (D) COUNTRY: Denmark (E) POSTAL CODE (2IR): 1871 (j) NAME OF THE INVENTION: Cytochrome-P450-monooxygenase(s) NUMBER OF SEQUENCES: 21 (m) COMPUTER READABLE FORM: (A) MEDIA TYPE: Diskette

(B) COMPUTER: IVM ROS (C) OPERATING SYSTEM: РО-ББО5/М5-605 (Г) PROGRAM: Raiepiip KeiIease y41.0, Megviop 21.25 (ERO) (2) INFORMATION FOR 5EO IO MO: 1 : () SEQUENCE CHARACTERISTICS: (A) LENGTH: 1929 base pairs (B) TYPE: nucleic acid (B) STRANDING: double (U) TOPOLOGY: linear (s) TYPE OF MOLECULE: cDNA (mi) DIRECT SOURCE: (B) CLONE : РА5ООХ(их) FEATURE: (A) NAME/LCOD: СО5 (B) LOCATION: 81..1673 (xi) SEQUENCE DESCRIPTION: 5EO IO MO: 1

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IV 1650 155 UYU

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MEPATAMY KKU ZHUMYKHA HE SEZAMTAMY UHA AND KRALUVSTMI Ua

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SKARKU TARY PETTY ISTUGITOT ZATASOTUMT VTRU KUDIH MTA And

SABLEULIKH VIEAMTVAV SOLIMUSASAS SIOJAUHT ALALALALIA LEVA IB

() SEQUENCE CHARACTERISTICS: (A) LENGTH: 531 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 2:

Mes Aza pu CHE Aza taye Rko S3r Tsyu ged sim S1u Bech chai ro oyi x 5 10 15 biz Ter NTK Sub iey Gez uUki Ted gech Rko Mai ieo io uv) bak 20: 25 39 tuk tus tem iem CHO bek olksh Beh Ako Av dho bet Aksh Beh Su uv

Tepy yiu 5iu Ata Rgo Ahu her Ro Rkesbiu Rgo Aza bip tey Rko T1e 55. 60 ivch s1u Ayep wa Niv Tl oien biu Ro Hey Rgoo Niv Pu5 Ayep Te Mo t 7 vo 1) gez Aza ysetsoAku Tuk Ziu Rgo Uvi! Me 14 Hey dku Izha u Teo na cho VE

UZE Rgo eat Uvi wai Mai back run LEFT fight Aza Aza Ato bi Mak Mo 150 105 110

Tuye Mai Ni Azr in Ayar o Suv Suv Zek Aud rko Ata Bek Ro siu rko 115 120 125

Gu Ah Iey Beh Tukh Avrorets Guz Ai Uvi S1u Re Aa Ro» Tukh Z1U 130. 135 140 siy oTuk TtroAgu Sino Mee Aku Puv Tets Be Ata tey biyu ikhah Tl Ve 145 150 155 o

Mes vk Vku Uki Buv Aa Azo Su Tukh Aza Ako bij Chip o C01y Meb dr i65 179 175

Aha rets Whoa! AtTa Ar Gey OR Akp Aza ATa Aza bek yub Aza Bek WHAT 150 185 1850 what

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Bo 255 - 275 hai Re Re ST: ruv Uvi 112 Ar bib Viv Meb Ayer Blo Div dru Ruo 275 280 285

Mah rRko AV Ap s1u biu v5r ey Uvi Avr Mai Tei yiye dep ei Sat 735 7235 399

Ace Ta Nib ABr o Siu Tk im Ako Be To: lk AvroNiya Uvi uv li 305 310 315 320 tTie vi iyev dar Tl Rve Tie S01u Ma 112 Ar Torg bek bek Uvi TVH

Zak 330 335 116 Pem o Tzhro dia Mer bek bi teo Mee dgo uv Rko bii Uai Bei hu 349 ЗИZ 350

Iuv Aza siy Aza siv Mai Ak vza Ma mah S1u Ar Avr Buv ro Ah 355 350 3655 chai At Beh Mr ABr div Via Puvo11e Bgo Tukh im Id Meb in Uvi 379. ZV 385 ihv bij Shg bez Dyud Tem NiB Ro Ro Hi Tvgoiiey iz Uai Rro ДЯ 285 350 395 400 fight Tvg Me Agro Avrobvh fight Tiz was 61Uu Tut AbBr o MA1 Rgo Via Ap 405 Fo 415

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BIV 50 - 525 lgh АТаА dia 530 (2) INFORMATION FOR 5EO IO MO: 3: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRENGTH: unknown (D) TOPOLOGY: linear ( i) TYPE OF MOLECULE: peptide

(m) FRAGMENT TYPE: M-terminal (mi) DIRECT SOURCE: (B) CLONE: M-terminal peptide (hi) SEQUENCE DESCRIPTION: 5EO IJUO MO: 3:

Av Tak Tyh Aa tlu Rgo bij ivy vy su (Chu Beg uv Re Act 1 5 10 15 (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 4: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (B) ORGANITY: unknown (D) TOPOLOGY: linear (s) TYPE OF MOLECULE: peptide (m) TYPE OF FRAGMENT: M-terminal (mi) DIRECT SOURCE: (B) CLONE: Internal peptide

Mei Ar. Ah cen Ma! Liz Avr ate Avri Ago Aa. Aa Div

I B 16 (2) INFORMATION FOR 5EO IO MO: 5: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 nucleotide pairs (B) TYPE: nucleic acid (B) STRANDING: single (G) TOPOLOGY: linear (y) TYPE MOLECULES: DNA (genomic) (mi) DIRECT SOURCE: (B) CLONE: Primer 1 (xi) SEQUENCE DESCRIPTION: ZEO AND MO: 6: aSaapAATTST TMPISSMOaA NMOMTT (2) INFORMATION FOR 5EO IO MO: 5: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) CHAIN STRENGTH: unknown (D) TOPOLOGY: linear (s) MOLECULE TYPE: peptide (m) FRAGMENT TYPE: internal (mi) DIRECT SOURCE: (B) CLONE: Amino acids encoded by primer 1 (xi) SEQUENCE DESCRIPTION: ZEO and MO: 6:

Rpe hav Rgo Z merda and (2) INFORMATION FOR 5EO IO MO: 7: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 nucleotide pairs (B) TYPE: nucleic acid (C) STRANDING: single (G) TOPOLOGY: linear (i) TYPE OF MOLECULE: DNA (genomic) (mi) DIRECT SOURCE: (B) CLONE: Primer 2 (xi) SEQUENCE DESCRIPTION: 5EO AND MO: 7

BSOOSATOST IYASANNOSK MOSCOW

(2) INFORMATION FOR 5EO IO MO: 8: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) CHAIN STRENGTH: unknown (D) TOPOLOGY: linear(s) MOLECULE TYPE: peptide (m ) FRAGMENT TYPE: internal (mi) DIRECT SOURCE: (B) CLONE: Amino acids encoded by primer 2 (hi) SEQUENCE DESCRIPTION: 5EO IU MO: 8 su Ago Ago Haa Suz hwa Siu 1 : B (2) INFORMATION FOR 5EO IU MO : 9: () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 pairs of nucleotides (B) TYPE: nucleic acid (B) STRENGTH: single (D) TOPOLOGY: linear

(i) TYPE OF MOLECULE: DNA (genomic) (mi) DIRECT SOURCE: (B) CLONE: 77 primer (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 9

AATASSASTO ASTATAE

(2) INFORMATION FOR 5EO IO MO: 10: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 nucleotide pairs (B) TYPE: nucleic acid (B) STRANDING: single (U TOPOLOGY: linear (s) MOLECULE TYPE: DNA ( genomic ) (mi) DIRECT SOURCE: (B) CLONE: gene-specific primer "12" (hi) SEQUENCE DESCRIPTION: 5EO IU MO: 10:

OSOSATOOVA STASTAVOOOOO TSOS

(2) INFORMATION FOR 5EO IO MO: 11: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 nucleotide pairs (B) TYPE: nucleic acid (B) STRANDING: single (U TOPOLOGY: linear (i) MOLECULE TYPE: DNA ( genomic) (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 11: TT TM ASSOCIATIONS (2) INFORMATION FOR 5EO IO MO: 12: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 nucleotide pairs (B) TYPE: nucleic acid (B ) STRENGTH: single (U TOPOLOGY: linear(s) TYPE OF MOLECULE: DNA (genomic) (mii) DIRECT SOURCE: (B) CLONE: gene-specific primer "7" (hi) SEQUENCE DESCRIPTION: 5EO IU MO: 12:

VSOLATSSOA SATSAASOOS SSO

2) INFORMATION FOR 5EO IO MO: 13: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 nucleotide pairs (B) TYPE: nucleic acid (B) STRANDING: single (U TOPOLOGY: linear (s) TYPE OF MOLECULE: DNA (genomic ) (mi) DIRECT SOURCE: (B) CLONE: Primer Z (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 13:

(2) INFORMATION FOR 5EO IO MO: 14: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 nucleotide pairs (B) TYPE: nucleic acid (B) STRANDING: single (U TOPOLOGY: linear (i) TYPE OF MOLECULE: DNA (genomic) (mi) DIRECT SOURCE: (B) CLONE: Primer 4 (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 14

SOSAASSTTA TTASATSTSA AOOSOBASSSS T

(2) INFORMATION FOR 5EO IO MO: 15: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 nucleotide pairs (B) TYPE: nucleic acid (B) STRANDING: single (U TOPOLOGY: linear (s) MOLECULE TYPE: DNA ( genomic) (mi) DIRECT SOURCE: (B) CLONE: Primer 5 (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 15:

(2) INFORMATION FOR 5EO IO MO: 16: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 nucleotide pairs

(B) TYPE: Nucleic acid (B) STRANDING: single (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: DNA (genomic) (mi) DIRECT SOURCE: (B) CLONE: Primer 6 (xi) SEQUENCE DESCRIPTION: 5EO I MO: 16

(2) INFORMATION FOR 5EO IO MO: 17: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 542 amino acids (B) TYPE: amino acid (C) CHAIN STRENGTH: unknown (D) TOPOLOGY: linear (y) TYPE OF MOLECULE : protein (ii) HYPOTHETICAL OPTIONS: NONE (her) ANTI-SENSE SEQUENCES: NONE (m) FRAGMENT TYPE: internal (m) SOURCE: (B) STRAIN: 5ipariv aira (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 17

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IMe Tyh Be RBe STU 030 sel Be tsuv Guz Meb My tuv chai Mai MeE 145 150 155 160

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Aza mes Re Yi Aia ie Su We Tuh Be ber Be was She Bek ABY 285 250 255.

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Mes dich der bek Beh Lia Yi1Te Meb Aer Buv Tukh Niv Abr Ro Te Te

Y 818 t5a 285 der ta Ato I1e Shuz Mes Ter dk sim Siu Buv pub TI bij H1E 253 285 300

Der re kem ler He name X16 beu XZe mue Avrobtu Bi OBU AzpobgOo 305 Zo 315 220 tem ila; Tzh Aza Ar 10 Tie Buv Web Tv iІ1e Buv ST Tyeb Ma) Mee 325 330 335

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For 0 45 350

Mek Mai Mep was Ruo sim Te 1eb Ak Guz Aza Meh siy STU I1e Ar 355 350 65

Akz mai MU siu yiv im Aho im Mah ZM bio Beh Avrodiv Bko uv 370 375 zvo

Im Vsp o Tukh Uai uv ALa 112 Gez Aku biz Div Re Ah Te Niv Ro 385 380 395 40 uv viv Aia Bre Depotei Rho Nie bai M1ia Gse bej Ver Aza ik Mai 805 0 415

Aia siu tTuk Ni She Rgo uv s1iu Vet pip o uv Tet ey Beh Aku Tut dgy 425 430 siu ich 01U ku bep orto Buv Ua) Ter D1a Asro Rgo Gets bek Be Buv 435 A4O 445

Vko sii Aku Ni Het Mem biz Sud Bek Si mah otet Bay oie iz lay 450 455 or

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BI 520 25 you eat Aku Eve Rgo Sat Ni Sat Tug Rgo Tag Ua! in 5ZO; ЗБ ДО (2) INFORMATION FOR 5EO IO MO: 18: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) CHAIN STRENGTH: single (D) TOPOLOGY: linear (s) TYPE OF MOLECULE: peptide (ii) HYPOTHETICAL OPTIONS: NONE (ii) ANTI-SENSE SEQUENCES: NONE (m) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: 5EO IYU MO: 18:

Guz Rio Si. Ago Ni Bey 1 5 (2) INFORMATION FOR 5EO IO MO: 19: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRENGTH: single (D) TOPOLOGY: linear (i) TYPE MOLECULES: peptide (ii) HYPOTHETICAL OPTIONS: NONE (ii) ANTI-SENSE SEQUENCES: NONE (m) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 19:

Te yu uv Aga siu was and 5 I (2) INFORMATION FOR 5EO IO MO: 20: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (C) STRENGTH: single

(D) TOPOLOGY: linear (s) MOLECULE TYPE: peptide (s) HYPOTHETICAL OPTIONS: NONE (s) ANTI-SENSE SEQUENCES: NONE (m) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: 5EO IU MO: 20: iuvlyago svid dgo Niv beu Ap li Suz Zeg Sish Ma! TNg Sam TVi 1 5 10 15

Avp Ar Mei Deu Rne Pe Zeg Re Zeg Tpg Ou was Aga Su Suz 95 30 . (2) INFORMATION FOR 5EO IO MO. 21: () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (B) CHAIN STRENGTH: single (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide (ii) HYPOTHETICAL OPTIONS: NONE (ii) ANTI-SENSE SEQUENCES: NONE (m) FRAGMENT TYPE: internal (hi) SEQUENCE DESCRIPTION: ZEO IU MO: 21: uv Rgo sim Aga Niv Re Avzp Siyu Suz beg spy Mai Tygoau Te sm i 5 10 15

Avp Ar vi Ago Re Me et Ripa. bug You are sleepy Aga Su Su 29 25 Zo Y

Claims (9)

1. A method of isolation of an enzyme that catalyzes the conversion of an aldoxime into a nitrile and the conversion of this nitrile into the corresponding cyanohydrin, which includes: (a) isolation and solubilization of microsomes from plant tissue that produces cyanogenic glycosides, (b) sending the solubilized microsomes into a column filled with with DEAE-Sepharose, washing the column and collecting the run and washing fractions, (c) identification of the fractions that contain the enzyme, (d) adding Tpup X-114 to the fractions obtained in step (c) and centrifugation until the formation of two phases, where the lower phase contains an enzyme. 2. The enzyme cytochrome P450ii-monooxygenase, which catalyzes the conversion of an aldoxime into a nitrile and the conversion of this nitrile into the corresponding cyanohydrin, which has a molecular weight of 55 kDa, which was determined by electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate (50O5-RACE), and M- the final sequence given in ZEO IYUO MO: 3, and obtained using the method according to claim 1. 3. Enzyme according to claim 2, which differs in that it has the sequence given in 5HEO IU MO: 2. 4. A DNA molecule that encodes an enzyme according to item 2 or 3. 5. The DNA molecule according to claim 4, which is characterized by the fact that it encodes an enzyme of a plant selected from the group that includes the species of bZogapyt, T/yoyisht, Iypit, Takhiv, Tgidiospip, Mappinoi, Atudaaishv, Rhypiz and cruciferous plants. 6. The DNA molecule according to claim 4, which differs in that it encodes (cytochrome P45Oskh) the monooxygenase of the plant 5ogupyt Bisoog. 7. A method for isolating a cDNA molecule that encodes an enzyme that catalyzes the conversion of an aldoxime to a nitrile and the conversion of this nitrile to the corresponding cyanohydrin, which includes: (a) isolation of the enzyme using the method according to claim 1, (b) optional determination of complete or partial the amino acid sequence of the enzyme obtained in step (a) and the construction of oligonucleotides, (c) probing the cDNA library of plant tissue that produces cyanogenic glycosides with the help of antibodies produced against the enzyme or oligonucleotides specific for DNA that encodes 4-15 amino acids of the specified enzyme sequence , and (d) isolation of clones from the cDNA library, where these clones include a cDNA molecule that encodes an enzyme that catalyzes the conversion of an aldoxime to a nitrile and the conversion of this nitrile to the corresponding cyanohydrin. 8. A method of obtaining a transgenic plant, which includes DNA stably integrated into its genome, which encodes an enzyme that catalyzes the conversion of an aldoxime to a nitrile and the conversion of this nitrile to the corresponding cyanohydrin, which includes: (a) introduction into a plant cell or tissue, which can be regenerated into a whole plant, DNA comprising a gene expressed in this plant that encodes a monooxygenase according to any of claims 2-3, or a DNA molecule according to any of claims 4-6, and (b) selection of transgenic plants , which contain the specified DNA. 9. A method of obtaining a transgenic plant that exhibits reduced expression of an enzyme that catalyzes the conversion of an aldoxime to a nitrile and the conversion of this nitrile to the corresponding cyanohydrin, which includes: (a) introducing DNA into a plant cell or tissue that can be regenerated into a whole plant, according to any of claims 4-6, which encodes a sense RNA, an antisense RNA or a ribozyme, the expression of which reduces the expression of the enzyme according to any of claims 2-3, and (b) selection of transgenic plants.