UA127176C2 - Herbicide-tolerant maize plant dbn9858, and nucleotide sequence and method for detecting same - Google Patents

Abstract

Provided are a nucleotide sequence for detecting a maize event DBN9858 existing in a biological sample, and a detecting method therefor.

Description

This application claims priority to Chinese patent application Mo 201510219911.8 filed on April 30, 2015 and entitled "Corn plant ОВМ9У9858 resistant to herbicide and nucleotide sequence and method for its detection", the content of which is hereby included, as a reference

FIELD OF THE INVENTION

The present invention relates to a nucleic acid sequence for the detection of herbicide-resistant maize plant ЮВМО858 and a method for its detection, and in particular to a glyphosate- and glufosinate-resistant maize plant OVMO858 and a method for detecting whether a biological sample contains a DNA molecule from a specific transgenic object of corn rvmov858.

PRIOR ART

M-phosphonomethylglycine, also called glyphosate, is a systemic, long-acting, broad-spectrum sterilizing herbicide. Glyphosate is a competitive inhibitor of phosphoenolpyruvate (PEP), which is a synthetic substrate of 5-enolpyruvylshikimate-3-phosphate (EPP5), and can inhibit the conversion of both substrates, PEP and 3-phosphoshikimate, into

B-enolpyruvylshishikimate-Z-phosphoshikimate catalyzed by EPZP5, thus blocking the synthetic pathway of shikimic acid, which is a synthetic precursor of an aromatic amino acid, and disrupting protein synthesis that causes the death of plants and bacteria.

Glyphosate resistance can be realized by expression of a modified ERBR5.

Modified ERBR5 shows lower affinity to glyphosate. As such, in the presence of glyphosate, ERBR5 retains its catalytic activity, and thus glyphosate resistance is achieved.

Maize (7ea tauz I.) is the main food crop in many regions of the world. For corn production, an important agrotechnical feature is resistance to herbicides, in particular, resistance to the herbicide glyphosate. The resistance of corn to the herbicide glyphosate can be obtained by expressing the gene for resistance to glyphosate (ERBR5, SR) in a corn plant using a transgenic method, for example, the transgenic object of corn MKbOZ, the transgenic object of corn MOM88017, etc.

Recently, the widespread use of glyphosate-resistant cultivation systems and

The increased use of glyphosate has led to the predominance of glyphosate-resistant weeds. When faced with glyphosate-resistant weeds or in regions that are becoming more difficult to control weed species, the farmer can compensate for the weakness of glyphosate by mixing with another herbicide or by alternately using another herbicide capable of controlling missed weeds. by Yanam

Glufosinate is a non-systemic, non-selective herbicide from phosphinothricin herbicides. It is primarily used for post-emergence control of annual or perennial broadleaf weeds, and it controls weeds by irreversibly inhibiting I-phosphinothricin (the active ingredient in glufosinate) on glutamine synthase (an enzyme required to detoxify ammonia in plants). Unlike glyphosate, which kills roots, glufosinate primarily kills leaves and can be translocated into the xylem of the plant by plant transpiration, and is between paraquat and glyphosate in terms of rate of action.

The enzyme phosphinothricin-M-acetyltransferase (PAT), isolated from 5igeriotusev, catalyzes the conversion of I-phosphinothricin into its inactive form by acetylation. A gene expressing PAT in a plant-optimized form has been used in soybean to confer resistance to the herbicide glufosinate, such as the soybean transgenic object A5547-127. Thus, the herbicide glufosinate, applied in combination with a marker of resistance to glufosinate, may be a non-selective method for effective control of glufosinate-resistant weeds.

Meanwhile, in the large-scale cultivation of insect-resistant transgenic maize, a small number of surviving insects/pests can develop resistance after breeding for several generations. Like non-insect-resistant transgenic corn, herbicide-resistant transgenic corn cultivated in a defined proportion with insect-resistant transgenic corn can slow the development of resistance in insects/pests.

It is known that the expression of exogenous genes within a plant is influenced by their position on chromosomes, which may be due to the fact that the structure of chromatin (such as heterochromatin) or transcriptional regulatory element (such as an enhancer) is located close to the integration site. Thus, it is usually necessary to screen a large number of transgenic objects to identify a marketable transgenic object (ie, an object in which the introduced gene of interest produces optimal expression).

For example, in plants and other organisms, it was observed that the level of expression of the introduced gene varies significantly from one object to another; there may also be differences in spatial and temporal expression patterns, such as differences in the relative expression of a transgene in different plant tissues, such that the actual expression profile may differ from the expression profile expected according to the transcriptional regulatory element in the construct introduced with the gene . Thus, as a rule, it is necessary to make hundreds of different transgenic objects and screen each individual object that has the expected level and profile of transgene expression in order to commercialize such objects. An object with the expected level and profile of transgene expression can be used to introduce the transgene into another genetic environment through the sexual crossing of individuals of different species by the generally accepted method of crossing. The progeny obtained by such hybridization may retain the trait of transgene expression as in the original transformant. With this strategy, it can be ensured that many cultivars will have robust gene expression, and these cultivars can be well adapted to local growing conditions.

It would be useful to be able to detect the presence of a particular event in order to determine whether the offspring of a sexual cross contains the gene of interest. In addition, a method to detect a specific event would also be useful for compliance with relevant regulations, for example, food products derived from recombinant crops require official approval and labeling before being placed on the market. The presence of the transgene can be detected by any well-known method of detecting polynucleotides, such as polymerase chain reaction (PCR) or DNA hybridization using polynucleotide probes. These detection methods are generally focused on commonly used genetic elements, such as promoters, terminators, marker genes, etc.

As a result, such methods may be useless in distinguishing between different events, particularly those produced using the same DNA construct, unless the sequence of the chromosomal DNA ("flanking DNA") adjacent to the inserted transgene DNA is known. Thus, at this time, a particular transgenic object is often identified by PCR with a pair of primers that overlap the junction between the inserted transgene and the flanking DNA, and specifically, a first primer containing the flanking sequence and a second primer that contains the insertion sequence.

ESSENCE

The purpose of this invention is to create a nucleic acid sequence for detecting the herbicide-resistant maize plant OVMO858 and a method for its detection, in which the transgenic object of maize OVMU9858 has better resistance to the herbicide glyphosate and the herbicide glufosinate, and the detection method can accurately and quickly detect , whether the biological sample contains a DNA molecule from a specific transgenic object of corn OVMO858.

To achieve the above-mentioned goal, the present invention relates to a nucleic acid sequence containing at least 11 consecutive nucleotides in 5EO IU MO: C or their complementary sequence, and/or at least 11 consecutive nucleotides in "EO IU MO: 4 or their complementary sequence.

Preferably, the nucleic acid sequence contains ZEO IU MO: 1 or its complementary sequence, and/or 5EO IO MO: 2 or its complementary sequence.

Additionally, the nucleic acid sequence contains ZEO IO MO: C or its complementary sequence, and/or 5EO IO MO: 4 or its complementary sequence.

In addition, the nucleic acid sequence contains хEO ЮЮ MO: 5 or a sequence complementary to it.

ZEO IO MO: 1 or its complementary sequence is a sequence 22 nucleotides in length located near the insertion junction at the 5' end of the insertion sequence in the maize transgenic object OVMU858, and encompassing the genomic DNA flanking sequence near the inserts and the DNA sequence at the 5' end of the insert sequence.

The inclusion of ZEO IU MO: 1 or its complementary sequence can be identified as the presence of the transgenic object of corn ЮОВМУО858. 5EO IU MO: 2 or its complementary sequence is a sequence 22 nucleotides in length located near the insertion junction at the 3'-end of the insertion sequence in the rvmov858 maize transgenic object, and spans the genomic DNA flanking sequence near the site insertions and sequence

DNA at the 3' end of the insertion sequence. The inclusion of ZEO I MO: 2 or its complementary sequence can be identified as the presence of the rvmov858 transgenic object of corn.

In the present invention, the nucleic acid sequence can be at least 11 or more consecutive nucleotides in any part of the sequence of the transgenic insert in the ZEO IU

MO: From or its complementary sequence (the first nucleic acid sequence), or 60 at least 11 or more consecutive nucleotides in any part of the 5' region from the flanking DNA of the maize genome in ZEO IU MO: From or its complementary sequence (the second nucleic acid sequence acids). The nucleic acid sequence can additionally be a part, homologous or complementary to the ZEO IU MO: 3 containing the complete ZEO IU MO: 1.

When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences can be used as a pair of DNA primers in a DNA amplification method to obtain an amplification product. When the amplification product produced by the DNA amplification method using a pair of DNA primers is an amplification product containing ZEO IO MO: 1, the presence of the transgenic object of corn ЮОВМУО858 or its offspring can be diagnosed. As is well known to those skilled in the art, the first and second nucleic acid sequences do not necessarily consist entirely of DNA, and may include RNA, a mixture of DNA and RNA, or a combination of DNA,

RNA or other nucleotides or their analogs that do not function as templates for one or more polymerases. Additionally, a probe or primer of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 contiguous nucleotides in length and may be selected from the nucleotide sequences provided in ZEO IU MO: 1, 5EO IU MO: 2, 5EO I MO: 3, 5EO IU MO: 4 and 5EO IUO MO: 5. When the probe and primer are selected from the nucleotide sequences proposed in 5EO IU MO: 3, ZEO IUO MO: 4 and 5EO IU MO: 5, they can be at least about 21 to about 50 or more consecutive nucleotides in length. 5EO IU MO: C or its complementary sequence is a sequence 1301 nucleotides in length located near the junction of the insert at

B-ends of the insertion sequence in the transgenic object of corn ЮОВМУО858. 5EBEO IJU MO: C or its complementary sequence consists of a flanking sequence of genomic DNA of maize, which is 1067 nucleotides long (nucleotides 1-1067 in 5EO IJU MO: 3), the sequence of the DNA construct OVM10006, which is 89 nucleotides long (nucleotides 1068-1156 in 5EO IU MO: 3) and the DNA sequence at the 5'-end of the pg355 promoter, which is 145 nucleotides in length (nucleotides 1157-1301 in 5EO IU MO: 3) Inclusion of ZEBEO IU MO: 3 or its complementary sequence can to be identified as the presence of the transgenic object of maize OVMO9858.

A nucleic acid sequence may contain at least 11 or more contigs

Of the nucleotides in any part of the sequence of the transgene insert in the 5EO IU MO: 4 or its complementary sequence (the third nucleic acid sequence), or at least 11 or more consecutive nucleotides in any part of the 3' region of the flanking genomic DNA of maize in the 5EO IU MO: 4 or its complementary sequence (fourth nucleic acid sequence). The nucleic acid sequence can additionally be a part, homologous or complementary to the 5EO IU MO: 4 containing the complete ZEO IUO MO: 2. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences can be used as a pair of DNA primers in the DNA amplification method to obtain the amplification product. When the amplification product produced by the DNA amplification method using a pair of DNA primers is an amplification product containing ZEO IU MO: 2, the presence of the transgenic object of corn OVMUO858 or its progeny can be diagnosed. 5EBEO AND MO: 4 or its complementary sequence is a sequence 1310 nucleotides in length located near the insertion junction at the 3'-end of the insertion sequence in the transgenic maize object YOVMU858. 5EO IUO MO: 4 or its complementary sequence consists of the terminator sequence of IMo5, which has 164 nucleotides (nucleotides 1-164 in BEO

IO MO: 4), the sequence of the DNA construct OVM10006, which has 84 nucleotides (nucleotides 165-248 in ZEO IO MO: 4), and the flanking sequence of genomic DNA, which has 1062 nucleotides, in the maize integration site (nucleotides 2449-1301 in 5EO IU MO: 4). The inclusion of ZEO IU MO: 4 or its complementary sequence can be identified as the presence of the transgenic object of corn OVMO9858.

ZEO IUO MO: 5 or its complementary sequence is a sequence 6890 nucleotides in length for characterizing the transgenic object of maize OVMO858 and, in particular, contains the genomes and genetic elements as shown in Table 1. Inclusion of 5EO IUO MO: 5 or its complementary sequence can be identified as the presence of the transgenic object of corn OVMO9858.

Table 1

Genomes and genetic elements in 5EO IU MO: 5 in 1111117 177711111171717171717891711717171717171717111711111111lobv-1561С vv 77777777777717171717171717117 17111717171717171717171784111111111111111111111115745-5828...:(SKZh:UZ

The nucleic acid sequence or its complementary sequence can be used in a DNA amplification method to obtain an amplicon, which is detected as a diagnosis of the presence of a transgenic object of corn OVMOU858 or its offspring in a biological sample; and the nucleic acid sequence or its complementary sequence can be used in a method of detecting nucleotides to detect the presence of the rvmo858 corn transgenic object or its progeny in a biological sample.

To achieve the above goal, the present invention additionally relates to a method for detecting the presence of DNA of a transgenic object of corn OVMO9858 in a sample, which includes: contact of the sample for detection with at least two primers in the nucleic acid amplification reaction; carrying out the nucleic acid amplification reaction; and detecting the presence of the amplification product; where the amplification product contains at least 11 consecutive nucleotides in "ES IUO MO: Z or its complementary sequence, or at least 11 consecutive nucleotides in KEO IU

MO: 4 or its complementary sequence.

In addition, the amplification product contains consecutive nucleotides in positions 1-11 or in positions 12-22 in 5BO IU MO: 1 or its complementary sequence, or consecutive nucleotides in positions 1-11 or in positions 12-22 in ZEO IU MO: 2 or its complementary sequence.

In addition, the amplification product contains ZEO AND MO: 1 or its complementary sequence 5EO AND MO: 2 or its complementary sequence, ZEO IU MO: 6 or its complementary sequence, or ZEO IU MO: 7 or its complementary sequence.

In the technical solutions described above, the primers contain at least one of the nucleic acid sequences.

Specifically, the primer comprises a first primer selected from ZEO IU MO: 8 and 5EO IU MO: 10, and a second primer selected from ZEO IU MO: 9 and ZEO IU MO: 11.

To achieve the above goal, this invention additionally belongs to the method for

From the detection of the presence of the DNA of the transgenic object of corn OVMOU858 in the sample, which includes: contact of the sample for detection with a probe containing at least 11 consecutive nucleotides from 5EO I MO: From or a sequence complementary to it, or at least 11 consecutive nucleotides from HEO IU MO: 4 or its complementary sequence; hybridization of the sample for detection with probes under strict hybridization conditions; and detecting hybridization of the detection sample with the probe.

Harsh conditions may include hybridization in a solution of bh55C (sodium citrate), 0.595 505 (sodium dodecyl sulfate) at 652C, followed by a single washing of the membrane 2x550, 0.195 505, and 1x5ZS, 0.195 505, respectively.

Additionally, the probe contains consecutive nucleotides at positions 1-11 or at positions 12-22 with

ZEO IUO MO: 1, or consecutive nucleotides in positions 1-11 or in positions 12-22 in 5EO IU

MO: 2.

In addition, the probe contains 5EO IU MO: 1 or its complementary sequence, cXEO IU MO: 2 or its complementary sequence, 5xEO IU MO: 6 or its complementary sequence, or

ZEO IU MO: 7 or its complementary sequence.

To achieve the above-mentioned goal, the present invention additionally relates to a method for detecting the presence of DNA of the transgenic object of maize OVMO858 in a sample, which includes: contact of the sample for detection with nucleic acid marker molecules containing at least 11 consecutive nucleotides from "FEO IU MO: C or its complementary sequence, or at least 11 consecutive nucleotides from ZEO IU MO: 4 or its complementary sequence; hybridization of the sample for detection with nucleic acid marker molecules under strict hybridization conditions; and detecting hybridization of the detection sample with marker nucleic acid molecules to determine whether glyphosate resistance and/or glufosinate resistance is genetically linked to the nucleic acid marker molecules by marker hybridization analysis.

Additionally, the marker nucleic acid molecules contain consecutive nucleotides in positions 1-11 or in positions 12-22 of 5EBO IU MO: 1, or consecutive nucleotides in positions 1-11 or in positions 12-22 of BEO IU MO: 2.

In addition, the nucleic acid marker molecules contain ZEBEO IU MO: 1 or its complementary sequence, zZEO IU MO: 2 or its complementary sequence, ZEO IU

MO: 6 or its complementary sequence or 5EBEO IO MO: 7 or its complementary sequence.

Optionally, at least one of the probes is labeled with at least one fluorescent group.

To achieve the above goal, the present invention additionally relates to a DNA detection kit containing at least a DNA molecule containing at least 11 consecutive nucleotides in a sequence homologous to 5EO IJUO MO: C or complementary to it, or at least 11 consecutive nucleotides in a sequence homologous to ZEO

IU0 MO: 4 or its complementary sequence, and is suitable as a DNA primer or probe specific to the transgenic object of maize OVMO858 or its progeny.

Additionally, the DNA molecule contains consecutive nucleotides in positions 1-11 or in positions 12-22 of 5EBO IU MO: 1 or its complementary sequence, or consecutive

From nucleotides in positions 1-11 or in positions 12-22 of BEO IO MO: 2 or its complementary sequence.

In addition, the DNA molecule contains a sequence homologous to 5EO IO MO: 1 or a sequence complementary thereto, a sequence homologous to 5EO I MO: 2 or a sequence complementary thereto, a sequence homologous to 5EO I MO: 6 or a sequence complementary thereto, or a sequence homologous to 5EO IU MO: 7 or its complementary sequence.

To achieve the above objective, the present invention further relates to a plant cell comprising: a nucleic acid sequence encoding a glyphosate resistance protein

EPZP5, the nucleic acid sequence encoding the glufosinate resistance protein PAT and the nucleic acid sequence in the defined region containing the sequence proposed in 5EO IJU MO: 1, 5EO IJU MO: 2, 5EO IJU MO: 6 or 5EO IJU MO: 7 .

To achieve the above objective, the present invention additionally relates to a method for obtaining a corn plant that has resistance to the herbicide glyphosate, which includes: introducing a nucleic acid sequence encoding a glyphosate resistance protein ERB5P5 and a nucleic acid sequence in a specified region in the genome of a corn plant, where the nucleic acid sequence in the defined region contains at least one nucleic acid sequence selected from the sequences proposed in 5EO IU MO: 1, 5EO IUO MO: 2, 5EO IU MO: 3, ZEO IU MO: 4, 5EO IU MO: 5, 5EO IYUO MO: 6 and BEO IYUO MO: 7.

Specifically, a method for producing a corn plant having resistance to the herbicide glyphosate comprises: sexually crossing a first parent corn plant of the rvmoev858 transgenic corn object with resistance to the herbicide glyphosate with a second parent corn plant without resistance to the herbicide glyphosate, to obtain a large number of plants - descendants; treatment of offspring plants with the herbicide glyphosate; and selection of glyphosate-resistant progeny plants.

To achieve the above-mentioned purpose, the present invention additionally relates to a method for obtaining a corn plant having resistance to the herbicide glufosinate, which includes: introducing a nucleic acid sequence encoding a protein of resistance to glufosinate PAT and a nucleic acid sequence in a defined area in the genome of a corn plant, where 60 the nucleic acid sequence in the specified region contains at least one nucleic acid sequence selected from the sequences proposed by 5EO IU MO: 1, 5EO IO MO: 2,

ZEO IU MO: 3, 5EO AND MO: 4, BEO IU MO: 5, 5EOIU MO: 6 and ZEO IU MO: 7.

Specifically, a method for producing a corn plant having resistance to the herbicide glufosinate includes: sexually crossing a first parent corn plant of the rvmov858 transgenic corn object with resistance to the herbicide glufosinate with a second parent corn plant without resistance to the herbicide glufosinate, to obtain a large number of plants - descendants; treatment of offspring plants with the herbicide glufosinate; and selection of glufosinate-resistant progeny plants.

To achieve the above object, the present invention further relates to a method for producing a corn plant having resistance to the herbicide glyphosate and the herbicide glufosinate, comprising the introduction of a nucleic acid sequence encoding a protein of resistance to glyphosate

EPZR5, a nucleic acid sequence encoding a protein of resistance to glufosinate PAT and a nucleic acid sequence in a specified region in the genome of a corn plant, where the nucleic acid sequence in the specified region contains at least one nucleic acid sequence selected from the sequences proposed by 5EO IU MO: 1, 5EO IO MO: 2,

ZEO IU MO: 3, 5EO IU MO: 4, BEO IYUO MO: 5, BEO IO MO: 6 and BEO IO MO: 7.

Specifically, the method for obtaining a corn plant having resistance to the herbicide glyphosate and the herbicide glufosinate includes: sexually crossing the first parent corn plant of the rvmoev858 transgenic corn object with resistance to the herbicide glyphosate and the herbicide glufosinate with a second parent corn plant without resistance to the herbicide glyphosate and/or glufosinate herbicide, to obtain a large number of offspring plants; treatment of offspring plants with the herbicide glyphosate and the herbicide glufosinate; and selection of progeny plants resistant to glyphosate and glufosinate.

To achieve the above objective, the present invention further relates to a method of cultivating a corn plant resistant to the herbicide glyphosate, which includes: planting at least one seed of corn containing in its genome a nucleic acid sequence encoding the glyphosate resistance protein ERBRB5, and the sequence nucleic acid in the specified area; growing corn seed to corn plant; spraying the corn plant with an effective dose of the herbicide glyphosate, and harvesting the plant with reduced plant damage compared to other plants lacking the nucleic acid sequence in the specified area; where the nucleic acid sequence in the specified area is at least one nucleic acid sequence selected from the sequences proposed in 5EO IYUO MO: 1,

ZEO IYU MO: 2, ZEO IYU MO: 3, ZEO IYU MO: 4, ZEO IYU MO: 5, ZEOIYUO MO: 6 and BEO I MO: 7.

To achieve the above objective, the present invention further relates to a method of cultivating a corn plant resistant to the herbicide glufosinate, which includes: planting at least one seed of corn containing in its genome a nucleic acid sequence encoding a protein of resistance to glufosinate PAT, and the sequence nucleic acid in the specified area; growing corn seed to corn plant; spraying the corn plant with an effective dose of the herbicide glufosinate, and harvesting the plant with reduced plant damage compared to other plants lacking the nucleic acid sequence in the designated area; where the nucleic acid sequence in the specified region is at least one nucleic acid sequence selected from the sequences proposed in 5EO IU MO: 1,

ZEO IYU MO: 2, ZEO IYU MO: 3, ZEO IYU MO: 4, ZEO IYU MO: 5, ZEOIYUO MO: 6 and BEO I MO: 7.

To achieve the above objective, the present invention additionally relates to a method of cultivating a corn plant that has resistance to the herbicide glyphosate and the herbicide glufosinate, which includes: planting at least one seed of corn containing in its genome a nucleic acid sequence encoding the glyphosate resistance protein ERBR5 , the nucleic acid sequence encoding the protein of resistance to glufosinate PAT, and the nucleic acid sequence in the specified region; growing corn seed to corn plant; spraying a corn plant with an effective dose of glufosinate herbicide and glufosinate herbicide, and harvesting the plant with reduced plant damage compared to another 60 plants lacking the nucleic acid sequence in the designated area;

where the nucleic acid sequence in the specified region is at least one nucleic acid sequence selected from the sequences proposed in 5EO IU MO: 1,

ZEO IYU MO: 2, ZEO IYU MO: 3, ZEO IYU MO: 4, ZEO IYUO MO: 5, ZEO IYUO MO: 6 and BEO IU MO: 7.

To achieve the above object, the present invention further relates to a method for protecting plants from damage caused by a herbicide, which includes applying a herbicide containing an effective dose of glyphosate and/or glufosinate to a field where at least one transgenic corn plant is planted, wherein the transgenic corn plant contains in its genome at least one nucleic acid sequence selected from the sequences proposed in ZEO IU MO: 1, ZEO IU MO: 2, 5EO IUO MO: 3, 5EO IO MO: 4, 5EO IO MO: 5, 5EO

IO MO: 6 and ZEO IO MO: 7, and the transgenic corn plant has resistance to the herbicide glyphosate and/or the herbicide glufosinate.

To achieve the above object, the present invention further relates to a method of controlling weeds in a field, which includes applying a herbicide containing an effective dose of glyphosate and/or glufosinate to a field where at least one transgenic corn plant is planted, wherein the transgenic corn plant contains in its genome at least one nucleic acid sequence selected from the sequences proposed in the ZEO IYUO MO: 1,

FAILURE MO: 2, 5EO IYUO MO: 3, 5BO IYU MO: 4, 5EBEO IYUO MO: 5, BEOIYUO MO: 6 and 5EO IYU MO. 7, and the transgenic corn plant has resistance to the herbicide glyphosate and/or the herbicide glufosinate.

To achieve the above object, the present invention further relates to a method for controlling glyphosate-resistant weeds in a field with glyphosate-resistant plants, comprising applying a herbicide containing an effective dose of glufosinate to a field planted with at least one transgenic plant corn resistant to glyphosate, where the transgenic corn plant resistant to glyphosate contains in its genome at least one nucleic acid sequence selected from the sequences proposed in 5ХЕО ІЮ MO: 1, ZEO ІЮО МО: 2,

ZEO IU MO: 3, 5EO IU MO: 4, ZEO IU MO: 5, ZEO IUO MO: 6 and 5EO IO MO: 7, and a transgenic corn plant resistant to glyphosate is also resistant to the herbicide glufosinate.

To achieve the above objective, the present invention further relates to a method for slowing the development of resistance in insects, which includes planting at least one transgenic corn plant with resistance to glyphosate and/or glufosinate in the field where the plant is planted

From corn with resistance to insects, where the transgenic corn plant with resistance to glyphosate and/or glufosinate contains in its genome at least one nucleic acid sequence selected from the sequences proposed in ZEO IU MO: 1, 5EO IUO MO: 2, 5EO IO MO : 3, 5EO IU

MO: 4, BEO IU MO: 5, BEO IU MO: 6 and BEO IU MO: 7.

To achieve the above-mentioned goal, the present invention additionally relates to an agricultural product or product containing a polynucleotide with ZEO IU MO: 1 or ZEO

IU MO: 2, where the agricultural product or commodity is corn meal, corn meal, corn oil, corn starch, corn gluten, corn meal, cosmetic preparation, or filler.

For the nucleic acid sequence for detecting a herbicide resistant corn plant and the method for detecting the sequence of the present invention, the following definitions and methods may better define the present invention and guide one skilled in the art in the practical implementation of the present invention. Unless otherwise specified, terms should be understood in accordance with the generally accepted usage of a specialist in the given field. "Corn" belongs to the 7ea tauch, and includes all varieties of plants that can cross with corn, including wild types of corn.

The terms "comprising" and "including" refer to "including as non-limiting examples".

The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell cultures and tissues from which a plant can be regenerated, plant calli, plant shoots and intact plant cells in plants or parts of plants, such as embryos, pollen , seed germ, seeds, leaves, flowers, shoots, fruits, stems, root, root tips, anthers, etc. It should be understood that a part of a transgenic plant within the scope of this invention, as non-limiting examples, belongs to plant cells, protoplasts, tissues, calli, embryos, as well as flowers, stems, fruits, leaves and roots, and plant parts mentioned above, which are obtained from transgenic plants or their descendants that were previously transformed by the molecule

DNA according to the present invention and thus at least partially consist of transgenic cells.

The term "gene" refers to a fragment of nucleic acid that expresses a specific protein, including regulatory sequences before (5'-coding sequences) and after (3'-coding sequences) the coding sequence. "Native gene" refers to a naturally occurring gene with its regulatory sequences. A "chimeric gene" refers to any gene that is not a native gene, including regulatory and coding sequences that do not occur together in nature. "Endogenous gene" refers to a native gene in its natural location in an organism's genome. "Exogenous gene" refers to a gene that is currently present but was originally absent from the organism's genome, and also refers to a gene introduced into a recipient cell by transgenic steps. An exogenous gene may contain a native gene or a chimeric gene introduced into a non-natural organism. "Gransgene" is a gene that was introduced into the genome by transformation. The site in the plant genome where the recombinant DNA has been introduced may be referred to as the "insertion site" or "target site". "Flanking DNA" can include a genome normally found in an organism, such as a plant, or exogenous (heterologous) DNA introduced by transformation, such as a fragment associated with a transformation event. Thus, the flanking DNA may contain a combination of native DNA and exogenous DNA. In the present invention, a "flanking region" or "flanking sequence" or "genomic border region" or "genomic border sequence" refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500 or 5000 base pairs or more, located immediately before or after the exogenous DNA molecule inserted first, and adjacent to the exogenous DNA molecule inserted first. When positioned to an insert, the flanking region may also be designated as "left flanking" or "3'-flanking" or "3'-genomic flanking" or "genomic 3'-flanking," etc. When positioned after the insertion, the flanking region may also be designated as "right flanking" or "5'-flanking" or "5'-genomic flanking region" or "genomic 5'-flanking sequence", etc.

Transformation methods that result in random integration of exogenous DNA will result in transformants containing different flanking regions that are characteristic and unique to each transformant. When recombinant DNA is introduced into a plant through conventional crossing, its flanking regions will generally not change. Transformants will also contain unique junctions between the insertion site of heterologous DNA and genomic DNA or two regions of genomic DNA or two regions of heterologous DNA. A "junction" is the point where two specific DNA fragments join. For example, splicing occurs when insert DNA joins flanking DNA. A junction also occurs in a transformed organism when two pieces of DNA are joined together in a manner modified from that found in the native organism. "Junction DNA" or "junction site" refers to DNA containing a junction point.

The present invention relates to the transgenic object of corn designated as ОВМО858 and its progeny, where the transgenic object of corn ЮОВМУ858 is a plant of corn rvmo858, including plants and seeds of the transgenic object of corn ОВМО9858, as well as its plant cells or parts, from which it may be recovered, including, but not limited to, cells, pollen, seed primordia, flowers, buds, roots, stems, pistils, inflorescences, cobs, leaves, and products from the OVMO858 corn plant, such as corn meal, corn fine flour, corn oil, liquid cornmeal and extract, corn stigmas, corn starch, and biomass remaining in cornfields.

The transgenic object of corn OVMO9858 according to the present invention contains a DNA construct, and if the construct is expressed in plant cells, the transgenic object of corn ОЯвВМ9858 produces resistance to the herbicides glyphosate and glufosinate. The DNA construct contains two expression cassettes in sequence. The first expression cassette contains a suitable promoter for expression in a plant, operably linked to the gene encoding 5-enolpyruvylshikimate-3-phosphate synthetase (ERBRB) with resistance to the herbicide glyphosate, and a suitable polyadenylation signal sequence. The second expression cassette contains a suitable promoter for expression in a plant, a functionally linked 3 gene encoding a phosphinothricin-M-acetyltransferase (PAT) with resistance to the herbicide glufosinate, and a suitable polyadenylation signal sequence. Additionally, the promoter may be a promoter isolated from a plant, including constitutive, inducible and/or tissue-specific promoters; a suitable promoter includes, but is not limited to, the Cauliflower mosaic virus (Samum) 355 promoter, the Morning mosaic virus (EMM) 355 promoter, the T511 promoter, the ubiquitin promoter, the actin promoter, the nopaline synthase (MO5) promoter, the Aadgobasiegsht shptegiasiep5 promoter, the octopine synthase (OS5) promoter, the yellow leaf curl Sevzigit, patatin promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (KiVizSO) promoter, glutathione 5-bo transferase promoter (051), E9U promoter, 505 promoter, aisA/aisA promoter, Kor promoter

Adhorasiegisht gpi7odepez and promoter 5isa2 Agaridorzib5. The polyadenylation signal sequence can be a suitable polyadenylation signal sequence that functions in a plant, including, but not limited to, a polyadenylation signal sequence derived from the nopaline synthase (MOB) gene Adgobasiepyut Shshptegasiep5, cauliflower mosaic virus (Samu) 355 terminator, E9U ribulose-1,5 terminator -bisphosphate carboxylase/pea oxygenase, protease inhibitor II gene (RIM I) and o-tubulin gene.

In addition, the expression cassette may additionally contain other genetic elements, including, but not limited to, an enhancer and a signal peptide/transit peptide. An enhancer can increase the level of gene expression, including, but not limited to, the tobacco engraving virus (TEM) translational activator, the Samm355 enhancer, and the EMMZ355 enhancer.

A signal peptide/transit peptide can direct the ERBRZ protein and/or the PAT protein to traffic outside the cell or to specific organelles or compartments within the cell, for example, targeting to the chloroplast by the sequence encoding the chloroplast transit peptide, or targeting to the endoplasmic reticulum by of the holding sequence "KOEI".

The 5-enolpyruvylshikimate-Z-phosphate synthetase gene (ERBR5) can be isolated and obtained from the strain of SPA Adgobasiegisht shptegasiep5 5r., and it can be modified by the polynucleotide encoding

ERBZR5, by means of codon optimization or other methods, in order to increase the stability and accessibility of the transcript in the transformed cell. The 5-enolpyruvylshikimate-3-phosphate synthetase gene (ERBR5B) can also be used as a selective marker gene. "Glyphosate" refers to M-phosphonomethylglycine and its salts, and treatment with "glyphosate herbicide" refers to treatment with any herbicide formulation containing glyphosate. The selection of the rate of use of a certain herbicide with glyphosate to obtain an effective biological dose is within the skill of the ordinary technical specialist in agriculture.

Treatment of a field containing plant materials derived from herbicide-resistant corn plants rvmo858 with any herbicide formulation containing glyphosate will control weed growth in the field without affecting the growth or production of plant materials derived from the herbicide- resistant plants of corn OVMO858.

The gene of the phosphinothricin M-acetyltransferase (PAT) enzyme isolated from Bigeriotuse5

Zo miidospgotodepev, catalyzes the conversion of I-phosphinothricin into its inactive form by acetylation, and thus gives the plant resistance to the herbicide glufosinate.

Phosphinothricin (RTC, 2-amino-4-methylphosphonobutyric acid) is an inhibitor of glutamine synthetase. RTS is a structural unit of the antibiotic 2-amino-4-methylphosphonyl-alanyl-alanine, the tripeptide of which (PTT) has the property of resistance to gram-positive bacteria and gram-negative bacteria, as well as resistance to the fungus Voiguii5 sipegea. The phosphinothricin M-acetyltransferase (PAT) gene can be used as a selective marker gene. "Glufosinate", also called "phosphinothricin", belongs to the ammonium 2-amino-4-

Hydroxy(methyl/phosphonyl|butanoate), and treatment with "herbicide glufosinate" refers to treatment with any formulation of herbicide containing glufosinate. Selection of the rate of use of a particular herbicide with glufosinate to obtain an effective biological dose is within the skill of the ordinary agricultural technician. Treatment of a field containing plant material derived from herbicide-resistant corn plants YOVMO858 with any herbicide formulation containing glufosinate will control weed growth in the field without affecting the growth or production of plant material derived from the herbicide- resistant plants of corn OVMOU858.

The DNA construct is introduced into a plant by a transformation method, including, but not limited to, Adgobasiegisht-mediated transformation, gene gun-mediated transformation, and pollen tube transformation.

Adhorasiegisht-mediated transformation is a common method for plant transformation. Exogenous DNA, which is going to be introduced into the plant, is cloned into the vector between the right and left border sequences, that is, T-DNA sections. Adhorasiegisht cells are transformed with the vector, which will then be used to infect plant tissue, which allows T-DNA vector sections containing exogenous DNA to be inserted into the plant genome.

Gene gun-mediated transformation is the bombardment of plant cells with a vector containing exogenous DNA (particle-mediated biolistic transformation).

Pollen tube transformation is when exogenous DNA is transferred via the natural pollen tube pathway (also called pollen tube transfer tissue) formed after pollination of the plant via the nucellus pathway into the blastocyst.

After transformation, the transgenic plant must be recovered from the transformed plant tissue, and the progeny carrying the exogenous DNA selected using a suitable marker.

A DNA construct is a combination of DNA molecules that are joined together to provide one or more expressing cassettes. The DNA construct is preferably a plasmid capable of self-replication within the bacterial cell and contains various restriction endonuclease recognition sites for insertion of DNA molecules that provide functional genetic elements, i.e., promoters, introns, leader sequences, coding sequences, 3'-terminator regions and other sequences. Expression cassettes included in the DNA construct contain the genetic elements necessary to ensure transcription of the template RNA and can be engineered for expression in prokaryotic or eukaryotic cells.

Most preferably, the expressing cassettes of the present invention are designed for expression in plant cells.

A transgenic "object" is obtained by transforming plant cells with a heterologous DNA construct, i.e., includes an expression cassette of nucleic acid containing the gene of interest, restoration of a population of plants obtained as a result of insertion by a transgenic method into the genome of a plant, and selection of a specific plant , characterized by an insertion at a specific location in the genome. The term "subject" refers to the original transformant and progeny of the transformant containing heterologous DNA. The term "subject" also refers to offspring obtained by sexual crossing between a transformant and an individual from another species containing heterologous DNA. Even after repeated backcrossing with the parent, the insert DNA and flanking genomic DNA from the transformant parent is also present in the progeny from the cross at the same location on the chromosome. The term "subject" also refers to the DNA sequence from the parent transformant containing the insert DNA and the flanking genomic sequence immediately adjacent to the insert DNA that is expected to be transmitted to progeny obtained by a sexual cross between one parental line containing the insert DNA (eg, the original transformant and the progeny obtained by

From self-pollination) and a parental line that does not contain the insert DNA and that receives the insert DNA with the gene of interest.

As used herein, "recombinant" refers to a form of DNA and/or protein and/or organism that is not normally found in nature and thus obtained by manual intervention. Such manual intervention may produce a recombinant DNA molecule and/or a recombinant plant. A "recombinant DNA molecule" is obtained by artificially combining two, otherwise separated, segments of sequences, for example, by chemical synthesis or by manipulating isolated nucleic acid segments through genetic engineering.

Technologies for manipulating nucleic acids are well known.

The term "transgenic" includes any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of a heterologous nucleic acid, including those transgenic that have been so altered in the first place, as well as progeny individuals obtained by sexual crossing or asexual reproduction from original transgenics. In the present invention, the term "transgenic" does not include changes in the genome (chromosomal or extrachromosomal) by conventional methods of plant breeding or natural events such as random pollination, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation. "Heterogeneous" in this invention refers to that first molecule, which, as a rule, is not found in the form of a combination with the second molecule in nature. For example, a molecule can be obtained from a first species and inserted into the genome of a second species. Thus, such a molecule is heterogeneous relative to the host and is artificially inserted into the genome of the host cell.

The transgenic maize object YVMOU858 with resistance to the herbicide glyphosate and the herbicide glufosinate is cultivated using the following steps: primary sexual crossing of the first parent maize plant with the second parent maize plant to produce a variety of first-generation progeny plants in which the primary parent maize plant consists of maize plants , which are cultivated from the rvmov858 transgenic object of corn and its descendants, which are obtained by transformation with the expressing cassette according to the present invention with resistance to the herbicide glyphosate and the herbicide glufosinate, and the second parent plant of corn lacks resistance to the herbicide glyphosate and the herbicide glufosinate; and then 60 selection of progeny plants with resistance to the use of the herbicide glyphosate and/or the herbicide glufosinate, which allows the cultivation of corn plants with resistance to the herbicide glyphosate and the herbicide glufosinate. These steps may further include backcrossing the progeny plant with resistance to the herbicide glyphosate and/or the herbicide glufosinate with a second parent corn plant or with a third parent corn plant; and then selection of the progeny by application of the herbicide glyphosate and/or the herbicide glufosinate or by detection of a molecular marker that correlates with the trait (such as a DNA molecule containing a junction region found at the 5'-end and 3'-end of the insertion sequence in the transgenic object of corn OVMO858), thus obtaining corn plants with resistance to the herbicide glyphosate and the herbicide glufosinate.

It should also be understood that two different transgenic plants can also be crossed to produce offspring containing two separate and separately added exogenous genes.

Self-pollination of suitable progeny can produce progeny plants homozygous for both added exogenous genes. Backcrossing with the parent plant and outcrossing with the non-transgenic plant as described above can also be assumed, and asexual propagation does the same.

Vi transgenic maize is capable of killing, for example, Hieridoriega insects/pests and

S. coli, but there are still a small number of surviving insects/pests that, after breeding for several generations, can possibly evolve into insects/pests resistant to Vi protein. In order to solve the problem of resistance development in insects/pests, the US Department of Environmental Protection offers the following guide for the use of transgenic crops: a certain proportion of shelter corn must be provided (shelter corn is needed in different quantities depending on the crop, being 595, 1095, 2095 and etc., and they can be transgenic corn plants without insect resistance (such as herbicide-resistant transgenic corn plants) or corn plants resistant to pests of no interest, and not necessarily transgenic corn plants).

After most of the insects/pests have been killed on the corresponding insect-resistant transgenic plants, some of the insects/pests will still survive on the shelter corn, allowing the non-resistant insect/pest population to dominate. Thus, even if a small number of resistant insects/pests survive, the resistance genes will be substantial

Zo bred after crossing with dominant insects/pests with no resistance.

The term "probe" refers to an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule, such as a radioactive isotope, ligand, chemiluminescent substance, or enzyme, is attached. Such a probe is complementary to the nucleic acid chain of interest, in the case of the present invention, the genomic chain

DNA from transgenic object of corn OVMO858, whether genomic DNA from transgenic object of corn ЮОВМОУ858 or seeds, or from plant, or seeds, or extract of transgenic object of corn 0ОВ8МУО858. Probes of the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a DNA sequence of interest and that can be used to detect the presence of a DNA sequence of interest.

The term "primer" refers to a portion of an isolated nucleic acid molecule that is annealed to the complementary DNA strand of interest by nucleic acid hybridization to form a hybrid between the primer and the DNA strand of interest and then extended along the DNA strand of interest. , using a polymerase, for example, DNA polymerase. A pair of primers according to the present invention relates to their use for amplification of nucleic acid sequences of interest, for example, by polymerase chain reaction (PCR) or other generally accepted methods of nucleic acid amplification.

Probes and primers are typically 11 or more polynucleotides in length,

BO preferably, 18 or more polynucleotides, more preferably, 24 or more polynucleotides, and most preferably, 30 or more polynucleotides. Such probes and primers specifically hybridize to the sequence of interest under high stringency hybridization conditions.

Preferably, the probes and primers of the present invention have complete sequence identity to the nucleic acid sequence of interest, although probes differing from the DNA sequence of interest that retain the ability to hybridize to the sequences

The DNA of interest can be engineered by conventional methods.

Primers and probes based on sequences of flanking genomic DNA and the insert according to the present invention can be confirmed by generally accepted methods, for example, by isolating the corresponding DNA molecules from plant material obtained from the transgenic object of corn YUVMU858, and determining the nucleic acid sequence of this DNA molecule.

The DNA molecule contains a transgenic inserted sequence and a flanking region of the maize genome, a fragment of which can be used as a primer or probe.

Nucleic acid probes and primers of the present invention hybridize under stringent conditions to the DNA sequence of interest. Any generally accepted method of hybridization or amplification of nucleic acids can be used to detect the presence of DNA from the YUOVMU858 transgenic object of corn in the sample. Nucleic acid molecules or their fragments are capable of specific hybridization with other nucleic acid molecules under certain conditions. As used herein, two nucleic acid molecules are capable of specifically hybridizing with each other if the two nucleic acid molecules are capable of forming an antiparallel, double-stranded nucleic acid structure. A nucleic acid molecule is "complementary" to another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules exhibit "full complementarity" when each nucleotide of one molecule is complementary to a nucleotide of another molecule. Two molecules are "minimally complementary" if they hybridize to each other with sufficient stability to allow them to anneal to each other at least under conventional "low stringency" conditions. Similarly, molecules are "complementary" if they hybridize to each other with sufficient stability to allow them to anneal to each other at least under conventional "high stringency" conditions. Deviations from complete complementarity are acceptable, provided that such deviations do not completely interfere with the ability of the two molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to be able to form a stable double-stranded structure when using a specific solvent and salt concentrations.

As used in the present invention, a homologous sequence is essentially a nucleic acid sequence capable of specifically hybridizing with the complementary strand of another nucleic acid sequence to which it is mapped under high stringency conditions. Suitable stringency conditions that promote DNA hybridization, such as treatment with 6b,0x sodium chloride/sodium citrate (55202) at about 452C, followed by

With washing 2.0х555С at 502С, well known to specialists in this field. For example, the concentration of salts in the washing step can be selected from a low hardness of about 2.0x55C at 5020 to a high hardness of about 0.2x552 at 502C. In addition, the temperature in the wash step can be increased from room temperature under low stiffness conditions, about 2220, to high stiffness conditions, up to about 6520. | temperature and salt concentration can be changed; and either temperature or salt concentration can be kept constant while the other variable is varied. Preferably, a nucleic acid molecule of the present invention will specifically hybridize with one or more nucleic acid molecules proposed in 5EO IUO MO: 1, 5EO IUO MO: 2, 5EO IUO MO: 3, 5EO IUO MO: 4, 5EO IUO MO: 5 ,

ZEO IU MO: 6 and 5EO IU MO: 7, or with sequences complementary to them, or any fragment of the above sequences under moderately strict conditions, for example, at approximately 2.0x5552; and about 6520. Additionally preferably, a nucleic acid molecule of the present invention will specifically hybridize to one or more nucleic acid molecules set forth in 5EO IU MO: 1, 5EO IUO MO: 2, 5EO IO MO: 3, 5EO I

MO: 4, 5EBEO IO MO: 5, 5BEO IJU MO: 6 and 5BO IJUO MO: 7, or with their complementary sequences, or any fragment of the above sequences under high stringency conditions. In the present invention, a preferred nucleic acid marker molecule has the nucleic acid sequence set forth in ZEO IU MO: 1, BEO IU MO: 2, 5EO IU MO: 3,

ZEBEO IYU MO: 4, 5EBEO IYU MO: 5, 5BO I MO: b and 5BO IYUO MO: 7 or sequences complementary to them, or any fragment of the above sequences. Another preferred nucleic acid marker molecule of the present invention has from 8095 to 10095, or from 9095 to 10095 sequence identity with "EO IU MO: 1, 5EO IU MO: 2, 5EO IO MO: 5, 5EO IUO MO: 4,

ZEO IYU MO: 5, BEO IYUO MO: 6 and 5BEO IYUO MO: 7, or with sequences complementary to them, or with any fragment of the above sequences. ZEO IU MO: 1, 5EO IO MO: 2, 5ZEO

IO MO: 3, 5EO IO MO: 4, 5BO IU MO: 5, 5BEO IU MO: 6 and 5EO IO MO: 7 can be used as markers for plant selection methods to identify the offspring of genetic crossing.

Hybridization of the probe to the DNA molecule of interest can be detected by any method well known to those skilled in the art, including, but not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels.

With respect to amplification of nucleic acid sequences of interest (e.g., by PCR) using specific amplification primers, "stringent conditions" are conditions that allow the primers to hybridize only to the nucleic acid sequence of interest in a thermal reaction. DNA amplification, in which a primer having a wild-type sequence (or complementary to it) corresponding to a nucleic acid sequence of interest is capable of binding to a nucleic acid sequence of interest and preferably produces a unique amplification product that is amplicon itself.

The term "specific binding to (a sequence of interest)" indicates that the probe or primer under stringent hybridization conditions hybridizes only to the sequence of interest in a sample containing the sequence of interest.

As used herein, "amplified DNA" or "amplicon" refers to the product of nucleic acid amplification from a nucleic acid molecule of interest that is part of a nucleic acid matrix. For example, in order to determine whether a corn plant was obtained by sexual crossing with the OVMU858 transgenic corn object of the present invention, or whether a corn sample collected from the field contains the OVMO858 transgenic corn object, or whether it contains a corn extract such as crude powder, powder or oil, transgenic maize object OVMO858, DNA isolated from a tissue sample or extract of a maize plant can be subjected to a nucleic acid amplification method using a pair of primers to obtain an amplicon that is diagnostic for DNA of the transgenic maize object YUVMU858 . A pair of primers includes a first primer derived from a flanking sequence in the plant genome adjacent to the site of the exogenous DNA insert and a second primer derived from the exogenous DNA insert. The amplicon has a length and sequence that are also diagnostic for the transgenic maize object YUVMUO858. The length of the amplicon can vary from the binding length of the primer pair plus one nucleotide base pair, preferably, about plus fifty nucleotide base pairs, more preferably, about plus two hundred fifty nucleotide base pairs, and most preferably, about plus four hundred five seventy nucleotide base pairs or more.

Optionally, a pair of primers can be obtained from flanking genomic sequences inserted on both sides of the DNA to obtain an amplicon containing the complete nucleotide sequence of the insert. One of the pairs of primers obtained from the genomic sequence of the plant can be located at a distance from the DNA sequence of the insert, and this distance can vary from one nucleotide base pair up to approximately two hundred and fifty nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers that may form in a thermal DNA amplification reaction.

Amplification of polynucleic acids can be carried out by any method of amplification of polynucleic acids known in the art, including polymerase chain reaction (PCR). Various methods of nucleic acid amplification are well known to those skilled in the art.

PCR-based amplification methods have been developed to amplify up to 22 kb. genomic

DNA up to 42 kb Bacteriophage DNA. These methods, as well as other methods of DNA amplification known in the art, can be used in the present invention. The insertion sequence is exogenous

The DNA and flanking sequence of the DNA from the transgenic maize object YUVMU9858 can be amplified from the genome of the transgenic maize object YUVMU858 using the provided primer sequences followed by standard DNA sequencing from PCR amplicon or from cloned DNA.

DNA detection kits based on DNA amplification methods contain DNA primers that specifically hybridize to the DNA of interest and amplify the diagnostic amplicon under appropriate reaction conditions. The kit may provide an agarose gel-based detection method or a variety of methods known in the art to detect a diagnostic amplicon. The present invention provides a set containing DNA primers that are homologous or complementary to any part of the region of the maize genome with BEO IUO MO: C or 5EO IO MO: 4, and homologous or complementary to any part of the transgene insertion region with BEO IUO MO: 5.

In particular, the pair of primers determined to be suitable for DNA amplification methods is 5EO IUO MO: 8 and 5EO IU MO: 9, which amplify a diagnostic amplicon homologous to the part from the 5'-transgene/genome region of the transgenic object of corn YVMOU858, where the amplicon contains ZEO IU MO: 1. Other DNA molecules as DNA primers can be selected from ZEO IU MO: 5.

The amplicon obtained by these methods can be detected using a variety of methods.

One such method is the analysis of genetic bits, in which the DNA oligonucleotide is designed to cover both the DNA sequence of the insert and the adjacent flanking sequence of genomic DNA. The oligonucleotide is immobilized inside the wells of the tablet for microtitration. After PCR-based amplification of the region of interest (using one primer in the insert sequence and one in the adjacent flanking genomic sequence), the single-stranded PCR product can be hybridized to an immobilized oligonucleotide and used as a template for a single-base extension reaction with DNA- polymerase and labeled ZaMTP, specific for the expected next base.

The results can be obtained by a method based on fluorescence or EGI5A. The signal indicates the presence of the insert/flanking sequence, thus indicating successful amplification, hybridization, and single-base extension.

Another method is a pyrosequencing method, where the oligonucleotide chain is designed to span the junction of the insert DNA sequence and the adjacent genomic DNA. The oligonucleotide chain is hybridized with a single-stranded PCR product from the region of interest (one primer in the insertion sequence and one in the flanking genomic sequence), and then incubated with DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-/!-phosphosulfate and luciferin Add aMTP separately and then measure the generated light signal. The light signal reflects the presence of the insert/flanking sequence, indicating successful amplification, hybridization, and extension by one or more bases.

The phenomenon of fluorescence polarization, as described in Spepp, et al., (daepote Vev. 9:492-498, 1999) is also a method that can be used to detect the amplicon of the present invention. To use this method, it is necessary to construct an oligonucleotide chain covering the DNA sequence of the insert and the adjacent junction with genomic DNA. The oligonucleotide is hybridized with a single-stranded PCR product from the region of interest (one primer in the insert DNA and one in the flanking sequence of the genomic

DNA) and incubated with DNA polymerase and fluorescently labeled dZaMmTR. Elongation by one base leads to the insertion of ChMTP, which can be measured by the change in polarization using a fluorimeter. The change in polarization reflects the presence of an insert/flanking sequence, indicating successful amplification, hybridization, and single-base extension.

Tadtap is described as a method for detecting and quantifying the presence of a DNA sequence and is fully presented in the instructions provided by the manufacturer. Briefly, an oligonucleotide probe for EKET is designed to cover the DNA sequence of the insert and the adjacent junction with the flanking genomic DNA. Probe for EKET and primers for PCR (one

Two primers in DNA inserts and one in the flanking genomic sequence) participate in a cyclic reaction in the presence of a thermostable polymerase and aMTP. Hybridization of the probe for

EKET will lead to cleavage and release of the fluorescent group from the quencher molecule in the EKET probe. The fluorescent signal reflects the presence of the insert/flanking sequence, indicating successful amplification and hybridization.

Suitable hybridization-based methods for detecting plant materials derived from the YUOVMO858 transgenic maize object additionally include Southern blot hybridization, Northern blot hybridization, and ip 5yi hybridization. In particular, a suitable method includes: incubating the probe and the sample, washing to remove the probe that has not bound, and detecting hybridization of the probe. The described method of detection depends on the type of marker attached to the probe. For example, a radioactively labeled probe can be detected by exposure and development of X-ray film, or an enzymatically labeled probe can be detected by the color change that occurs when the substrate is converted.

Molecular beacons have been described for use in sequence detection as described in Tuapoi, et al. (Maishge Bioesp. 14:303-308, 1996). Briefly, an oligonucleotide probe for EKET is designed to cover the DNA sequence of the insert and the adjacent junction with the flanking genomic DNA. The unique structure of the probe for EKET contains a secondary structure that keeps the fluorescent and quenching molecules in close proximity. The probe for EKET and primers for PCR (one primer in the insert DNA and one in the flanking genomic sequence) participate in a cyclic reaction in the presence of a thermostable polymerase and amMTP. After successful PCR-based amplification, hybridization of the EKET probe with the target sequence results in loss of secondary structure by the probe and spatial separation of the fluorescent and quencher molecules, which creates a fluorescent signal. The fluorescent signal reflects the presence of the insert/flanking sequence, indicating successful amplification and hybridization.

Other described methods, such as microcurrent devices, offer a method and devices for the isolation and amplification of a DNA sample. Photodyes are used to detect and analyze a specific DNA molecule. A nanotube device suitable for detecting a DNA molecule according to the present invention contains an electronic sensor for detecting a DNA molecule or a nanobead that binds to a specific DNA molecule, and thus, 600 detection can be made.

DNA detection kits can be developed using the composition described in this invention and methods described or known in the field of DNA detection. The kit facilitates the detection of the presence of DNA from the transgenic maize object OVMOU858 in the sample, and can additionally be used for crossing maize plants containing DNA from the transgenic maize object OVMOU858. The kit may contain DNA primers or probes that are homologous or complementary to at least part of ZEO IU MO: 1, 2, 3, 4 or 5, or other DNA primers or probes that are homologous or complementary to the DNA contained in the transgenic DNA genetic elements , and these DNA sequences can be used as primers in amplification reactions

DNA or as probes in a DNA hybridization method. The structure of the DNA sequence of the transgenic insert and the connection with the genome of corn, included in the genome of corn, are shown in figure 1, and table 1 contains the flanking genomic region from the corn plant ЮОВМО858, located at the 5'-end of the sequence of the transgenic insert; part of the insertion sequence from the left border area (IB) from Adgobrasiegisht; the first expression cassette consisting of the Cauliflower mosaic virus 355 promoter (rg355) containing tandem repeats of an enhancer region functionally linked to glufosinate-resistant phosphinothricin M-acetyl transferase (cRAT) with 5igeriotusev5 and the Cauliflower mosaic virus 355 terminator (1355); a second expression cassette consisting of the rice actin-1 promoter (pHO5AcSiI) functionally linked to the sequence encoding the chloroplast transit peptide from Agaridorsis (Shaiyapa for ERBR5 (5rAiSTRa), with glyphosate-resistant 5-enolpyruvyl-3-phosphoshikimate synthase (SERBR5) from the cСР4 strain

Adgobrasiegisht, and with the terminator of nopaline synthase transcription (IMo5); part of the insertion sequence from the right border area (CB) from Adgobasiegisht; as well as the flanking genomic region from the maize plant OVMU858, located at the 3-end of the transgene insertion sequence (ZEO IU

MO:5). DNA molecules suitable as primers in DNA amplification methods can be obtained from any part of the sequence of the transgenic insert in the OVMO858 maize plant, as well as from any part of the DNA region from the flanking maize genome in the OVMO858 transgenic maize object.

The transgenic object of corn ЮОВМУО858 can be combined with other varieties of transgenic corn, for example, with corn resistant to herbicides (such as 2,4-О0, dicamba, etc.), or varieties of transgenic corn that carry other genes for resistance to insects (such as StutAb, MmirzA and

From t.p.). Crossing various combinations of these different transgenic objects with the transgenic maize object ЮОВМУО858 of the present invention can produce improved hybrid transgenic maize varieties which are resistant to various insects and herbicides and which can show more outstanding characteristics such as increased yield etc. .p., in comparison with non-transgenic varieties and transgenic varieties with one trait.

The present invention relates to a nucleic acid sequence suitable for detecting a herbicide-resistant corn plant OVMO858 and to a method of its detection, where the transgenic object of corn OVMO858 is resistant to the phytotoxic effect of agricultural herbicides containing glyphosate and/or glufosinate. Such a double-characterized maize plant expresses glyphosate-resistant proteins 5-enolpyruvylshishikimate-3-phosphate synthetase (ERBR5Z) from strain CP4A

Adhorasiegichts, which give the plant resistance to glyphosate; and glufosinate-resistant phosphinothricin M-acetyltransferase (PAT) proteins from 5igeriotuses, which confer resistance to glufosinate in the plant. Dual trait corn has the following advantages: 1) the ability to use agricultural herbicides containing glyphosate on corn crops to control a wide range of weeds; 2) the glufosinate resistance trait can act as a non-selective method effective for controlling glyphosate-resistant weeds when used in combination with glufosinate herbicides (mixed with glyphosate herbicides or alternatively); 3) herbicide-resistant transgenic corn, as transgenic corn without insect resistance, can slow down the development of insect/pest resistance when planted in a defined ratio with transgenic corn with pest resistance; 4) no decrease in corn yield. In addition, the genes encoding the traits of resistance to glyphosate and glufosinate are associated with the same DNA segment and are present in the same locus of the genome of the OVMU858 transgenic object of corn. This provides improved cross-breeding efficiency and allows tracing the fragment of the transgenic insert contained in reproductive populations and their progeny using a molecular marker. At the same time, ZEO IO MO: 1, 2, b or 7, or their complementary sequences can be used in the detection method according to the present invention as DNA primers or probes for obtaining amplification products, which are defined as the transgenic object of corn OVMO858 or its progeny , and quickly, accurately and stably detect the presence of plant materials obtained from the OVMU858 transgenic object of maize. 60 BRIEF DESCRIPTION OF SEQUENCES

ZEOIIUO MO: 1 11 nucleotides on both sides of the insertion site from the 5'-fragment of the transgene and genomic DNA of maize in the transgenic object of maize ravmo858;

ZEOIU MO: 2 11 nucleotides on both sides of the insertion site from the 3Z'-fragment of the transgene and genomic DNA of maize in the transgenic object of maize ravmo858;

ZEOIUO MO: C sequence of 1301 nucleotides in length, located next to the insertion junction site at the 5'-end of the insertion sequence in the transgenic object of maize OVMO9858;

ZEOIIUO MO: 4 sequence of 1310 nucleotides in length, located adjacent to the insertion junction site at the 5'-end of the insertion sequence in the transgenic object of maize OVMO858;

ZEOIUO MO: 5 complete sequence of T-DNA, 5'- and 3'-flanking sequences of the maize genome;

ZEOIUO MO: 6 sequence located inside 5XEO IJUO MO: C, covering the sequence of the DNA construct ОВМ10006 and the sequence of the pg355 promoter;

ZEOIYUO MO: 7 sequence located inside ZEO IYU MO: 3, covering the sequence of the terminator yMoz and the sequence of the DNA construct ravm10006;

ZEO IU MO: 8 first primer for amplification ZEO IUO MO: 3;

ZEOIU MO: 9 second primer for amplification of ZEO IO MO: 3;

ZEOIU MO: 10 first primer for amplification of HEO IU MO: 4;

ZEO IUO MO: 11 second primer for amplification of ZEO IUO MO: 4;

ZEO IU MO: 12 primer on the 5'-flanking genomic sequence;

ZEOIU MO: 13 primer located on T-DNA paired with 5EO IU MO: 12;

ZEO IO MO: 14 primer on the 3'-flanking genomic sequence paired with ZEO IU

MO: 12, to determine the homozygous or heterozygous state of the transgene;

ZEOIIUO MO: 15 primer located on T-DNA paired with 5EO IYU MO: 14;

ZEOIIUO MO: 16 primer 1 for detection of EPZR5 by Tadtap;

ZEO IYUO MO: 17 primer 2 for detection of EPZR5 by Tadtap;

ZEOIU MO: 18 probe 1 for detection of EPOR5 by Tadtap;

ZEO IU MO: 19 primer C for detection of RAT by Tadtap;

ZEO IU MO: 20 primer 4 for detection of RAT by Tadtap;

ZEO IU MO: 21 probe 2 to detect RAT by Tadtap;

ZEO IU MO: 22 first primer for endogenous corn ubiquitin gene;

ZEO IU MO: 23 second primer for endogenous corn ubiquitin gene;

ZEO IYUO MO: 24 probe for RAT for detection of hybridization by Southern blotting;

ZEO IJU MO: 25 probe for EPOR5 for detection of hybridization by Southern blotting;

ZEO IU MO: 26 primer located on T-DNA, in the same direction as 5ZEO IU MO: 13;

ZEO AND MO: 27 primer located on T-DNA, in the opposite direction from 5EO IU

MO: 13, used to obtain the flanking sequence;

ZEO IU MO: 28 primer located on T-DNA, in the opposite direction from 5EO IU

MO: 13, used to obtain the flanking sequence;

ZEO IU MO: 29 primer located on T-DNA, in the same direction as ZEO IU MO: 15;

ZEO AND MO: 30 primer located on T-DNA, in the opposite direction from 5EO IU

MO: 15 used to obtain the flanking sequence;

ZEO IU MO: 31 primer located on T-DNA, in the opposite direction from 5EO IU

MO: 15 used to obtain the flanking sequence.

Further in this document, the technical solutions according to this invention will be further described in detail with reference to the accompanying drawings and examples.

BRIEF DESCRIPTION DRAWING

Figure 1 is a schematic diagram of the sequence of the transgene insert and the sequence of the connecting nucleic acid from the genome of corn, and the method of their detection according to the present invention, which is used to detect the corn plant OVMOU858, resistant to the herbicide.

Figure 2 is a schematic diagram of the nucleic acid sequences of the recombinant expressing vector OVM10006 and the method of its detection according to the present invention, which is used to detect the herbicide-resistant corn plant OVMO858.

DETAILED DESCRIPTION

Further in this document, the technical solutions for the nucleic acid sequence and its detection method according to the present invention, which are used to detect the corn plant rvmoev858, resistant to the herbicide, will be further illustrated with the help of specific examples.

Example 1. Cloning and transformation 1.1. Vector cloning

Recombinant expressing vector OVM10006 was constructed using a standard method of gene cloning (as shown in Fig. 2). Vector YOVM10006 contained two tandem expressing cassettes with a transgene. The first expression cassette consists of the Cauliflower mosaic virus 355 promoter (rg355) containing tandem repeats of an enhancer region functionally linked to the glufosinate-resistant phosphinothricin M-acetyl transferase (cRAT) from zgeriotuses and the Cauliflower mosaic virus 355 terminator (1355 ). The second expressing cassette consists of the rice actin promoter (pHO5AcSiI), functionally linked to the sequence encoding the chloroplast transit peptide from Agaridorzi5 Ipayap for ERBR5 (5rAISTR2), with glyphosate-resistant 5-enolpyruvyl-3-phosphoshikimate synthase (SERBRB). from strain

SRA Adhorasiegiit, and with the transcription terminator of nopaline synthase (IMo5).

Vector YOVM10006 transformed I BA4404 Adgobrasiyeegisht (Ipmigodep, Spisado, O5A; catalog No. 18313-015) using the method with liquid nitrogen, and selection of transformed cells was carried out with the help of 5-enolpyruvylshishikimate-3Z-phosphate synthetase (ERBRB) as a selective marker. 1.2. Transformation of plants

Transformation was carried out using the generally accepted method of infection

Adhorasiegisht Maize germ was cultivated under sterile conditions from Adgobrasiegisht,

As described in part 1.1 of this example, for the transfer of T-DNA in the constructed recombinant expressing vector ОВМ10006 into the genome of corn, thus obtaining the transgenic object of corn ОВМО858.

Regarding the generally accepted method of transformation of corn, mediated

Adhorasiegisht: in short, immature embryos were isolated from corn and brought into contact with a suspension

Adhorasiegisht, where the nucleotide sequences of ERBRZ and PAT genes could be transferred to at least one cell of one of the embryos with the help of Adhorasiegisht (stage 1: infection stage). At this stage, the embryos were mainly immersed in a suspension of Adgobasiegicht (OOvvo-0.4-0.6, medium for infection (salt M5 4.3 g/l, vitamin M5, casein 300 mg/l, sucrose 68.5 g/l, glucose 36 g/L, acetosyringone (AB) 40 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-0) 1 mg/L, pH 5.3) to initiate inoculation Embryos were co-cultured with Adgobasiegiit for a period of time (3 days) (stage 2: co-cultivation stage). Preferably, the embryos after the infection stage were cultured on a solid medium (salt M5 4.3 g/l, vitamin M5, casein 300 mg/l, sucrose 20 g/l, glucose 10 g/l, acetosyringone (AB) 100 mg/l, 2,4-dichlorophenoxyacetic acid (2,4-O)) 1 mg/l, agar 8 g/l, pH 5.8). After this period of joint cultivation, an optional stage of "restoration" was foreseen. At the "recovery" stage, there was at least one antibiotic known to inhibit the growth of Adgobasiegisht (a cephalosporin) in the recovery medium (salt M5 4.3 g/l, vitamin M5, casein 300 mg/l, sucrose 30 g/l, 2.4 - dichlorophenoxyacetic acid (2,4-O) 1 mg/l, phytagel 3 g/l, pH 5.8), but no selective agent for transformant plants was added (stage 3: recovery stage). Preferably, the embryos are cultured on a solid medium with an antibiotic, but without a selective agent, to remove Adgobasiegisht and provide a recovery period for the infected cells. Next, the infected embryos were cultured on a medium containing a selective agent (glyphosate) and selected by growing transformed callus (stage 4: selection stage). Preferably, the embryos were cultivated on a solid medium for selection with a selective agent (salt M5 4.3 g/l, vitamin M5, casein 300 mg/l, sucrose 30 g/l, M-«(phosphonomethyl)glycine 0.25 mol/l , 2,4-dichlorophenoxyacetic acid (2,4-Y)) 1 mg/l, phytagel 3 g/l, pH 5.8), obtaining selective growth of transformed cells. The calli were then regenerated into plants (stage 5: regeneration stage).

Preferably, the calli that grew on the medium containing the selective agent were cultured on a solid medium (M5 medium for differentiation and M5 rooting medium) 60 to restore the plant.

Resistant calli that passed the screening were transferred to M5 medium for differentiation (M5 salt 4.3 g/l, vitamin M5, casein 300 mg/l, sucrose 30 g/l, b-benzyladenine 2 mg/l, M- (phosphonomethyl )uglycin 0.125 mol/l, phytagel 3 g/l, pH 5.8), and cultivated at 252C for differentiation. Differentiated seedlings were transferred to M5 medium for rooting (salt M5 2.15 g/l, vitamin M5, casein 300 mg/l, sucrose 30 g/l, indole-3-acetic acid 1 mg/l, agar 8 g/l, pH 5.8), were cultivated at 252C to a height of about 10 cm, and then transferred to a greenhouse and cultivated until they became robust. In the greenhouse, cultivation was carried out at 282C for 16 hours daily, followed by 202C for 8 hours. 1.3. Identification and screening of transgenic objects

A total of 332 individual transgenic To plants were obtained.

The presence of ERBR5 and RAT genes in restored transgenic maize plants was detected using TadMap' analysis (see example 2), and the number of copies in lines resistant to glyphosate and glufosinate was characterized. Through screening, it was found that object OVMU9858 was excellent and was characterized by one gene copy, good resistance to glyphosate herbicides, resistance to glufosinate herbicides and agronomic traits (see example 5).

Example 2. Detection of transgenic object of corn OVMU9858 using TadMap

Approximately 100 mg of leaves were taken as a sample from the transgenic maize object YOVMO858, genomic DNA was isolated using the OMeabvu Riapi Mahi kit from Oiadep, and the copy number of ERBRZ and RAT genes was detected by fluorescent quantitative PCR with the Tadtap probe. At the same time, wild-type corn plants were used as a control, and detection and analysis were performed as described above. Experiments were performed in three repetitions and the average was taken.

The specific method was as follows:

Step 11. Approximately 100 mg of leaves were taken as a sample from the rvmo858 transgenic object of corn and homogenized in a mortar using liquid nitrogen. Each sample was taken in three repetitions;

Stage 12. Genomic DNA from the above samples was isolated using the OMease kit

Riapi Mahi from Oiadep. For a specific method, please refer to the product instructions;

Stage 13. The concentration of genomic DNA of the above samples was determined using

MapoOhor 2000 (Tpegto Zsiepiyis);

From Stage 14. The concentrations of genomic DNA of the above samples were adjusted to the same concentration value in the range of 80-100 ng/μl;

Step 15. The copy number of the above samples was detected by fluorescent quantitative PCR with a Tadtap probe, a sample with a known copy number was used as a standard, and a wild-type maize plant was used as a control. Each sample was made in three repetitions and the average was taken. Primers and probes for fluorescent quantitative PCR were, respectively:

The following primers and probes were used to detect the sequence of the EPOR5 gene:

Primer 1: STOSADASOSOASSASOSTSATSAATA as proposed in 5EO IYU MO: 16 from the sequence list;

Primer 2: ТООСООССАТТОСОдДААТСОАОС as proposed in 5EO IЙО MO: 17 from the sequence list;

Probe 1: АТССАСОСОАТОСОССОСССССОТА as proposed in ZEO IU MO: 18 from the sequence list;

The following primers and probes were used to detect the sequence of the PAT gene:

Primer 3: SASTTOASATTAOOSSASSSTASAS as proposed in 5EO IO MO: 19 from the sequence list;

Primer 4: ТТГСАСАСТАСАСОТСТСААТОТААТОО as proposed in ZEO IYUO MO: 20 from the sequence list;

Probe 2: САССТОАТАТОоСсСОоСОотТТТТТОotТо as proposed in 5EO IU MO: 21 from the sequence list;

System for PCR reaction:

Each primer/probe mixture contains 45 μl of each primer at a concentration of 1 mM, 50 μl of 100

MCM of the probe and 860 μl of 1xTE buffer, and stored in an opaque tube at 426.

PCR reaction conditions:

Sir times

The data were analyzed using the software 5052.3 (Arriley Viosuziettv) and the transgenic object of corn OVMO858 with one copy was obtained.

Example 3. Identification of transgenic object of corn OVMO858 3.1. Isolation of genomic DNA

DNA was isolated according to the traditionally used method with STAV (hexadecyltrimethylammonium bromide): 2 g of young leaves from the OVMO858 transgenic object of corn were taken and ground into powder in liquid nitrogen. After that, 0.5 ml of buffer with STAB for DNA isolation, heated at 652C (20 g/l STAB, 1.4 M Mass, 100 mM Tris-HCl, 20

MM EDTA (ethylenediaminetetraacetic acid), pH adjusted to 8.0 with Mmaon), was thoroughly mixed and then extracted at 652C for 90 minutes. 0.5 volume of phenol and 0.5 volume of chloroform were added, mixed by inversion and centrifuged at a rotation speed of 12,000 rpm. (rpm) for 10 minutes. The supernatant was collected and 2 volumes of absolute ethanol were added. The centrifuge tube was gently shaken and left at 49C for 30 minutes. Centrifugation was performed at a rotation speed of 12,000 rpm. again for 10 minutes, and the DNA was precipitated to the bottom of the test tube.

The supernatant was removed, and the sediment was washed with 1 ml of ethanol with a mass concentration of 70,965, centrifuged at a rotation speed of 12,000 rpm. for 5 minutes and dried to dryness under vacuum or dried on an ultra-clean table. The DNA precipitate was dissolved in the appropriate amount of TE buffer (10 mm Tris-HCII, 1 mm EDTA, pH 8.0) and stored at a temperature of 20260. 3.2. Analysis of DNA flanking sequence

The concentration of the above DNA samples was evaluated, and the concentration of the tested samples was brought to the range of 80-100 ng/μl. Genomic DNA was cleaved with the help of selected restriction endonucleases Watni, Khta, Krp, zasi! (for the analysis of the 5'-end) and 5re, P5ii, Eso57I (for the analysis of the 3'-end), respectively. 26.5 μl of genomic DNA, 0.5 μl of the above selected restriction endonucleases and 3 μl of digestion buffer were added to each system for enzymatic digestion. Enzymatic cleavage was carried out for 1 hour. After completion of cleavage, 70 μl of absolute ethanol was added to the system for

From enzymatic cleavage, kept in an ice bath for 30 minutes, and centrifuged at a rotation speed of 12,000 rpm. within 7 minutes. The supernatant was removed and dried. After that, 8.5 μl of twice-distilled water (dango), 1 μl of 10x buffer for Ta were added. and 0.5 μl of Ta4-ligase and ligated at a temperature of 42C overnight.

Amplification by PCR was carried out using a series of nested primers for isolation of 5'- and 3'-trasgenic/genomic DNA. Specifically, a combination of DNA primers for isolation

B'-trasgenic/genomic DNA contains BEO IU MO: 13 Ii BEO IU MO: 26 as first primer, 5EO IO

MO:27 and ZEO IU MO:28 as second primer, and BEO IO MO:13 as sequencing primer. Combination

DNA primers for isolation of 3'-trasgenic/genomic DNA contain 5EO IU MO: 15 ii ZEO IU MO: 29 as first primer, 5EO IU MO: 30 ii BEO IU MO: 31 as second primer, and 5EO IU MO: 15 as sequencing primer. PCR reaction conditions are shown in Table 3.

The obtained amplicon was electrophoresed on a 2.095 agarose gel to isolate the PCR product, and then the fragments of interest were isolated from the agarose matrix using the OIAdiisk se! (Cat. Mo 28704, Oiadep Ips., MaIepsia, SA). After that, the purified PCR product was sequenced (for example, AVI RgiztTM 377, RE Viozuvietv, Eovieg Su,

SA) and analyzed (for example, with the Yuomabtak software,

OMA5TAV Ips., Maadizop, UM). The 5- and 3'-flanking sequence and the connecting sequence were determined using a standard PCR method. The 5'-flanking sequence and the junction sequence can be determined using ZEO IUO MO: 8 or 5EO IU MO: 12 in combination with ZEO IU MO: 9, 5EO IU

MO: 13 or 5EO IO MO: 26. The 3'-flanking sequence and the junction sequence can be determined using 5EO IUO MO: 11 or 5EO IO MO: 14 in combination with BEO IU MO: 10, 5EO I

MO: 15 or ZEO IU MO: 29. The system for the PCR reaction and the amplification conditions are shown in Tables 2 and

3. Those skilled in the art will understand that other primers can also be used to determine the flanking sequence and the connecting sequence.

DNA sequencing of the PCR product provides DNA suitable for the construction of other DNA molecules that will be used as primers and probes to detect corn plants or seeds derived from the OVMOU858 transgenic corn object.

It was found that the maize genomic sequence shown as nucleotides 1-1067 with BEO IO MO: 5 flanks the right border of the insertion sequence from the transgenic maize object OVMO858 (5'-flanking sequence), and the maize genomic sequence shown as nucleotides 5829- 6890 with 5EO IU MO: 5, flanks the left border of the insertion sequence from the transgenic object of maize YUBMO858 (3'-flanking sequence). The 5' junction sequence is shown in

ZEO IU MO: 1, and the 3'-connecting sequence is shown in 5ZEO IU MO: 2. 3.3. PCR test for zygosity

Linker sequences are relatively short polynucleotide molecules that are novel DNA sequences and are diagnostic of transgenic maize DNA

OVMU858, if they are detected in the analysis of the detection of several nucleic acids.

The connecting sequences in ZEO IU MO: 1 and 5EO IU MO: 2 represent the insertion site of the transgenic fragment in the transgenic object of corn OVMU858 and 11 polynucleotides of genomic DNA of corn on each side. Longer or shorter polynucleotide linker sequences can be selected from 5EO I MO: C or 5EO IU MO: 4. Linker sequences (5' junction region 5EO IO MO: 1 and 3' junction region 5EO IU MO: 2) are suitable as DNA probe molecules or DNA primers for DNA detection methods. The connecting sequences ZEBEO IO MO: b ii 5EBEO IO MO: 7 are also new DNA sequences in the maize transgenic object OVMO9858, which can also be used as DNA probe molecules or DNA primers to detect the presence of DNA of the maize transgenic object OVMO858.

ZEO IUO MO: 6 (from nucleotide 1068 to nucleotide 1301 5EO IUO MO: 3) covers the sequence

DNA constructs of ОВМ10006 and the sequence of the рг355 promoter, and 5EO ИХО MO: 7 (from nucleotide 1 to nucleotide 248 5EO ИО МО: 4) covers the sequence of the Моз terminator and the sequence of the DNA construct ОВМ10006.

Additionally, an amplicon is obtained using at least one primer with ZEO IU

Zo MO: Z ii ZEO IU MO: 4, and the primer produces a diagnostic amplicon for the transgenic maize object OVMO9858 when used in a PCR method.

Specifically, the PCR product is derived from the 5'-end of the transgene insert sequence, and the product

The PCR is part of the genomic DNA flanking the 5'-end of the T-DNA insertion sequence obtained from the genome of the plant material of the transgenic object of maize YUVMO858. This PCR product contains 5EO IO MO: 3. For amplification by PCR, design primer 5 (ZEO IU MO: 8) that hybridizes to the genomic DNA sequence flanking the 5'-end of the transgenic insertion sequence and paired primer b (5EBO IU MO: 9), located on the transcription termination sequence of transgenic IMo5.

The PCR product is obtained from the 3'-end of the sequence of the transgenic insert, and the PCR product contains part of the genomic DNA flanking the 3'-end of the T-DNA insert sequence obtained from the genome of the plant material of the transgenic object of maize YUVMUO858. This PCR product contains ZEO IO MO: 4. For PCR amplification, primer 8 (ZEO IO MO: 11) is designed to hybridize to the genomic DNA sequence flanking the 3'-end of the transgenic insert sequence, and primer pair 7 (ZEO IO MO : 10) from the sequence of the pg355 promoter located on

From the end of the insert.

The DNA amplification conditions shown in Table 2 and Table 3 can be used in the above PCR test for zygosity to obtain a diagnostic amplicon of the O8MU858 transgenic object of corn. Detection of amplicons can be carried out using a thermal cycler 5bigaiadepe Korosusieg, MO Epdipe, Regkip-EIteg 9700, or Errepaogiya Mabviegsusieg

BO Sgadieepi, as shown in Table 3, or using methods and devices known to those skilled in the art.

Table 2

PCR steps and reaction mixture conditions for the detection of the 5'-transgenic insert/genomic junction region of the transgenic object of maize OVMOU858. 4 Nuclease-free water Add to the final volume 20 μl of 1x final 2 10x reaction buffer (from 20 μl buffer concentration,

MaSiG) ' 1.5 mM final concentration of MaSiG» 10 mM solution of dATP, aStR, 200 μM final

With 0.4 μl concentration of each astrRiattrv amMtR

Primer 5 for a transgenic object (ZEO IO MO:8), 4 resuspended in 1x 02 μl of 0.1 μM final TE buffer or in water without 'nuclease concentration to a concentration of 10

MKM

Primer 6 for a transgenic object (ZEO IO MO:9), resuspended in 1x 02 μl of 0.1 μM final TE buffer or in water without 'nuclease concentration to a concentration of 10

MKM

RNase, free from DNases.

DNA polymerase KeoTazd 1.0 μl (recommended. 7 change pipettes before 1 unit/reaction (1 unit/μl) the next step

Isolated DNA (matrix): . letters from samples for the analysis of 200 ng of genomic DNA

Negative control. non-trasgenic corn

Negative control No DNA template (solution in which the DNA was resuspended) ng of genomic DNA

Positive control of corn containing rvmov858

Table C

Conditions for RegkKip-EITeg 9700 thermal cycler

Cycle number 942С From a minute 9420 30 seconds for 642 30 seconds 7226 1 minute 722 10 minutes

Gently mixed, if necessary (when there is no hot lid on the thermal cycler) 5 you can add 1-2 drops of mineral oil to the surface of each reaction liquid. PCR was performed on a thermocycler Bigaiadepe Korosusieg (Zigatadepe, Ha doMya, SA), MOU Epdipe (M K-Viogaad,

Negsshez, SA), Retkip-EIteg 9700 (Regkip EIteg, Vob5iop, MA) or Errepaogi Maviegsusieg Stadiepi (Errepaogi, Natbygod, Segtapu) using the above cycle parameters (table 3).

Thermal cycler Mu Epdipe or Errepdoghi Mabhiegsusieg Sgadiepi must work in calculation mode. The RegKip-EIteg 9700 thermal cycler operated at the maximum rate of temperature rise.

Experimental results show that when primers 5 and 6 (5ZEO IO MO: 8 and 9) were used in PCR reactions for genomic DNA of transgenic object of maize OVMO858, they led to an amplification product in the form of a 1301 bp fragment, and when they were used in PCR reactions for genomic DNA of untransformed maize and genomic DNA of maize that is not YUBMO858, no fragment was amplified; and when primers 7 and 8 (ZEO IYUO MO: 10 and 11) in PCR reactions for genomic DNA of the transgenic object of maize OVMOU858, they led to an amplification product in the form of a fragment of 1310 bp, and when they were used in PCR reactions for genomic DNA of untransformed maize and genomic DNA of maize that is not OVMO858, no fragment was amplified.

A PCR-based zygosity test can also be used to detect whether material obtained from the OVMU858 transgenic maize object is homozygous or heterozygous. Primer 9 (ZEO IO MO: 12), primer 10 (ZEO IU MO: 13) and primer 11 (ZEO IU

MO: 14) was used in the amplification reaction to obtain a diagnostic amplicon for the transgenic object of corn OVMOU858. The DNA amplification conditions shown in Table 4 and Table 5 can be used in the above zygosity test to obtain a diagnostic amplicon for the OVMO858 transgenic maize object.

Table 4

Reaction solution for the zygosity test. 2x Opimegza! Mavieg Mich (Arriiei 1x final 2 Viosuzietv, catalog number 5 μl x final 4304437 concentration

Primer 9 (5EO IU MO:12), primer 10 (5EO IU MO:13) and .

With primer 11 (ZEO IU MO:14) 0.3 μl 0.1 μM final and . concentration (resuspended in nuclease-free water to a concentration of 10 µM) 4 VEOTad DNA polymerase (1 1.0 µl (recommended 1 unit /µl) change pipettes before unit/reaction next step)

Extracted DNA (matrix): sheets of 200 ng genomic DNA from samples for analysis 50 ng genomic DNA non-transgenic positive control

B corn - Without DNA matrix (a solution in which

Negative control and resuspended DNA) - 50 ng of genomic DNA of corn, which

The positive control contains OVMO858

Table 5

Conditions for the ReikKkeep-EIteg 9700 thermal cycler for the zygosity test

Cycle number 952 10 minutes 10 9520 15 seconds 6490 1 minute (-12С/cycle) 9520 15 seconds 5420 1 minute 102С immersion

PCR was performed on a thermal cycler 5igaiadepe Kobosusieg (5igaiadepe, Ga XdoPa, SA), M Epdipe (MO V-Viogad, Negsciez, SA), RegKip-EIteg 9700 (RegKip EIteg, Vo5iop, MA) or Errepaogiya

Mahiegsusieg sgadiepi (Errepaogi, Natrigo, Seppapu) using the above cycle parameters (table 5). Thermocycler M.) Epdipe or Errepdogi Mabhiegsusieg Sgadiepi should work in calculation mode. Thermocycler Regkip-EIteg 9700 worked at the maximum rate of temperature rise.

In the amplification reaction, the biological sample from the matrix DNA contains DNA for diagnosing the presence of the transgenic object of corn ЮОВМУ858 in the sample. Alternatively, the reaction will contain two different DNA amplicons produced by a biological sample containing DNA derived from the maize genome. The DNA obtained from the maize genome is heterozygous for the corresponding insert DNA alleles present in the transgenic maize object OVMO858. These two different amplicons will correspond to the first amplicon obtained from the locus of the wild-type maize genome and the second amplicon to diagnose the presence of the DNA of the transgenic maize object YuVMU858. If only one amplicon corresponding to the second amplicon is obtained from the maize DNA sample, as described for the hybrid genome, the presence of the transgenic maize object ЮОВМОУ858 in the sample can be diagnosed and confirmed, and the sample was produced from maize seed that was homozygous for the corresponding of DNA alleles of the insert present in the OVMO858 transgenic object of maize.

It should be noted that primer pairs from the OVMU858 transgenic maize object were used to obtain amplicons that were diagnostic for genomic DNA of the OVMO858 transgenic maize object. These primer pairs include, but are not limited to, primers 5 and 6 (ZEO IO

MO: 8 and 9) and primers 7 and 8 (ZEO IO MO: 10 and 11) for use in the DNA amplification method.

Additionally, control primers 12 and 13 (5EO IU MO: 22 and 23) were included to amplify endogenous maize genes as an internal standard for reaction conditions. Analysis of the DNA extract of samples from the transgenic object of corn ЮОВМО858 must include a positive control from the extract of DNA from the tissue of the transgenic object, a negative control from the extract of DNA from tissue not from the transgenic object of corn ОВМО9858 and a negative control containing no matrix from the extract Maize DNA. In addition to these primer pairs, you can also use any primer pairs with ZEO IO MO: C or 5EO IO MO: 4, or their complementary sequence. When

Since they will be used in the DNA amplification reaction, they will accordingly produce amplicons containing

ZEO IJU MO: 1 or 5EO IJU MO: 2, which are diagnostic for tissues derived from the transgenic maize plant YOVMU858. The DNA amplification conditions shown in Tables 2 to 5 can be used to obtain diagnostic amplicons for the rvmo858 transgenic maize object with appropriate primer pairs. A DNA extract from a maize plant or seed or a product obtained from the transgenic maize object ЮВМОУ858 that produces diagnostic amplicons for the transgenic maize object ОВМО858 when tested by the amplification method

DNA, and which approximately contains the transgenic object of corn OVMOU9858, can be used as matrices for amplification to determine whether or not the transgenic object of corn rvmov858 is present.

Example 4. Detection of the transgenic object of corn OVMU858 using Southern blot hybridization 4.1. DNA isolated for Southern blot hybridization

Homozygous objects of transformation from the TA and T5 generations were used for Southern blot analysis. Approximately 5 to 10 g of plant tissue was ground in liquid nitrogen using a mortar and pestle. The plant tissue was resuspended in 12.5 ml of extraction buffer A (0.2 M Tris, pH 8.0, 50 mM EDTA, 0.25 M Mass, 0.195 vol/vol p-mercaptoethanol, 2.596 wt/vol polyvinyl -pyrrolidone), and centrifuged for 10 minutes at 4,000 rpm. (2755 9).

After removing the supernatant, the sediment was resuspended in 2.5 ml of extraction buffer B (0.2 M Tris, pH 8.0, 50 mM EDTA, 0.5 M Mass, 195 vol./o06. p-mercaptoethanol, 2.595 mab./0b . polyvinylpyrrolidone, 395 parcosyl, 2095 ethanol) and incubated at 37"C for 30 minutes. During incubation, the sample was mixed once with a sterile loop. After incubation, an equal volume of chloroform/isoamyl alcohol (24:11) was added, mixed gently by inversion and centrifuged for 20 minutes at 4,000 rpm. The aqueous layer was collected and 0.54 vol of isopropanol was added, followed by centrifugation for 5 minutes at 4,000 rpm to pellet the DNA. The supernatant was removed and the DNA pellet was resuspended in 500 µl TE To knock down any RNA present, DNA was incubated at 372C for 30 min with 1 µl 30 mg/ml RNase A, centrifuged for 5 min at 4,000 rpm and pelleted by centrifugation at 14,000 rpm ./min for 10 minutes in the presence of 0.5 volume of 7.5

M of ammonium acetate and 0.54 volume of isopropanol. After removing the supernatant, the precipitate was washed

500 μl of 7095 (mass fraction) of ethanol, and allowed to dry before dissolving in 100 μl of TE buffer. 4.2. Cleavage by restriction enzymes

DNA was quantified using a spectrophotometer or fluorimeter (using 1x TME and Hoechst stain).

Each time, 5 μg of DNA was cleaved in 100 μl of the reaction system. Genomic DNA was cleaved with the restriction enzymes zas and Nipap, respectively, and the partial sequence of ЕР5Р5З and РАТ in tТ-

DNA as a probe. Cleavage reactions were incubated overnight at the appropriate temperature for each enzyme. The samples were rotated in a high-speed vacuum to reduce the volume to 30 μl. 4.3. Gel electrophoresis

Bromophenol blue staining dye was added to each sample obtained in this example 4.2, and each sample was applied to a 0.795 agarose gel containing ethidium bromide. Electrophoretic separation was performed in TVE electrophoresis buffer and gel electrophoresis was performed at 20 volts overnight.

The gel was washed in 0.25 M NOSI for 15 minutes to depurine the DNA and then washed with water. The Southern blot was collected as follows: 20 sheets of thick dry filter paper were placed in a tray and 4 sheets of thin dry filter paper were additionally placed on top. One sheet of thin filter paper was pre-moistened with 0.4 M

Mmaon and placed on top of the paper stack, followed by a sheet of membrane to transfer Nuropa-

Ma (Ategopath RNagtasia Vioyesp, ЯКРМЗОЗВ), also soaked in 0.4 M Mahon. The gel was placed on top, ensuring that there were no air bubbles between the gel and the membrane. Three additional sheets of pre-wetted filter paper were placed on top of the gel and filled with 0.4 M Mahon buffer tray. The gel stack and buffer tray were connected using a wick pre-soaked in 0.4 M Mahon, and the DNA was transferred to the membrane. DNA transfer took approximately 4 hours at room temperature. After transfer, the Nurope membrane was washed in 2x 5Z5C for 10 seconds, and the DNA was bound to the membrane by UV cross-linking. 4.4. Hybridization

The appropriate DNA sequence was amplified by PCR to obtain probes. DNA-

The probes were ZEO IYU MO: 24 and 5EO IYUO MO: 25, or their homologous or complementary sequences. 25 ng of DNA probe in 45 μl of TE was boiled for 5 minutes, placed on ice for 7 minutes, and then transferred to a tube of Keaiiirgite I! (Ateg5pat RNaptasia Vioyesp, ZKRM1633).

After adding 5 μl of aSTtTP, labeled with ZR, to the Keairgitte tube, the probe was incubated at 3720 for 15 minutes. The probe was purified by centrifugation through a 05-50 microcentrifuge column (Ateg5Ppat RNaptasia Violiesny, y27-5330-01) according to the manufacturer's instructions to remove unincorporated αMTP. The activity of the probe was measured using a scintillation counter.

Nubrop membrane was pre-hybridized by soaking it in 20 ml of pre-heated Church solution for pre-hybridization (5200 mM MazRO, 1 mM EDTA, 795 505, 195 VZA) at 652C for 30 minutes. The labeled probe was boiled for 5 minutes and placed on ice for 10 minutes. An appropriate amount of probe (1 million pulses per 1 ml of pre-hybridization buffer) was added to the pre-hybridization buffer, and hybridization was performed at 652C overnight. The next day, the hybridization buffer was removed and after washing with 20 ml of 1 Cherch washing solution (40 mM MazRO, 1 mM EDTA, 595 505, 0.595 VZA), the membrane was washed with 150 ml of 1 Cherch washing solution at 652C for 20 minutes. This process was repeated twice with Church's washing solution 2 (40 mM MazRO, 1 mM EDTA, 195 505). The membrane was exposed to a fluorescent screen or X-ray film to reveal where the probe bound.

Three controls were included in each Southern blot: (1) DNA from a negative (untransformed) isolate used to detect any endogenous maize sequence that could hybridize with the element-specific probe; (2) DNA from a negative isolate that had been injected with OVM10006 cleaved by Nipa PI-dideiide in an amount equivalent to one copy based on the length of the probe to show the sensitivity of the experiment to detect a single-copy gene in the maize genome; and (3) plasmid OVM10006 cleaved by Nipa IS, which was equivalent to one copy based on the length of the probe, and which was used as a positive control for hybridization to show the sensitivity of the experiment.

The hybridization data provided strong evidence in support of PCR analysis with TadMap, meaning that the OVMOU9858 maize plant contains a single copy of the ERBR5 and RAT genes. Single bands of approximately 4 kb and 11 kb in size were obtained with this ERBRZ probe. respectively, upon enzymatic cleavage of zas I and Nipa I; single bands of approx.

3.5 t.p.n. and 1.8 t.p.n. respectively, during the enzymatic cleavage of ZAS I and Nip II. This indicates that ERBR5 and RAT were present in the transformed object of maize OVMO858 in one copy.

Example 5. Detection of resistance to herbicides in a transgenic object

The herbicide Koshypadyr (an aqueous solution of 4195 isopropylammonium glyphosate) and the herbicide Vavia (the active ingredient is glufosinate 1895) were used in this spray test.

A random location of the plots was used, carried out in three repetitions. The area was 15 m: (5 m x 3 m), the distance between lines was 60 cm, and the distance between plants was 25 cm, with generally accepted cultivation and care. There was a wide isolation zone of 1 m between the plots. Transgenic object of corn OVMO9858 was processed as follows: 1) without spraying; 2) spraying with Koshipair herbicide at the UZ leaf stage at a dose of 1,680 g a.i./ha (a.i./ha belongs to the "acid equivalent of the active ingredient per hectare"), then sprayed again with the same dose of Koipdir herbicide at the M8 stage; 3) spraying with herbicide

Vazia at the UZ leaf stage at a dose of 800 g a.i./ha (a.i./ha belongs to the "active ingredient per hectare"), then sprayed again with the same dose of Vavia herbicide at the U8 stage. It should be noted that different contents and dosage forms of the herbicide glyphosate can be converted into equal amounts of glyphosate acid, and different concentrations of glufosinate solution can be converted into equal amounts of the active ingredient glufosinate, all applicable to the following conclusions.

The phytotoxic symptom was examined 1 week and 2 weeks after application, respectively, and the corn yield of the plot was measured at harvest. The classification of phytotoxic symptoms is shown in Table 6. The degree of herbicide damage was used as an indicator to assess the resistance of transformants to herbicides, in particular, the degree of herbicide damage (95) U; (NUMBER of damaged plants of the same level x level number)/(total number of plants x highest level); where the degree of herbicide injury includes the degree of injury by glyphosate and the degree of injury by glufosinate, and the degree of herbicide injury was determined based on the results of a phytotoxicity study 2 weeks after treatment with glyphosate or glufosinate. Corn yield in each plot was measured by weighing the total yield (mass) of corn grains from three rows in the center of the plot. Differences in yield between different treatments were measured in the form of percent yield, percent yield (95)-yield with spraying/yield without spraying. The results of resistance of the transgenic object of corn

From rvmo858 to herbicide and corn yield results are shown in Table 7.

Table 6

Classification standards for the level of phytotoxicity of a herbicide relative to corn

The level of phytotoxicity - Description of the symptoms of yield or lack of yield

Table 7

The results of resistance of the transgenic object of corn OVMO858 to the herbicide and the results of the corn yield

Ron pinni vni ni tyny spraying) damage by glyphosate(90)d HER 77777777771111111111101111111c1 (Degree of damage by glufosinate(9b)) HER 7777777777771701111111111111111111c1C

Yield percentage (95) (glyphosate)

Percent yield (95) (glufosinate)

The results show that relative to the degree of herbicide damage (glyphosate and glufosinate) 1) the degree of damage to the transgenic object of corn OVMO9858 was essentially zero after treatment with the herbicide glyphosate (1680 g a.i./ha); and the degree of damage to the transgenic object of corn OVMU9858 was also essentially zero after treatment with the herbicide glufosinate (800 g a.i./ha); thus, the transgenic object of corn ЮОВМОУ858 has good resistance to herbicides (glyphosate and glufosinate).

Regarding yield: transgenic maize object OVMU9858 had no significant differences in yield after three treatments: no spraying, herbicide glyphosate (1680 ha.i./ha) and herbicide glufosinate (800 g a.i./ha); after spraying with the herbicide, the yield of the transgenic object of corn OVMO858, on the contrary, slightly increased, thus further indicating that the transgenic object of corn 0OVMO858 has good resistance to herbicides (glyphosate and glufosinate).

Example 6

Products such as agricultural products and goods can be obtained with the help of the transgenic object of corn YUVMUO858. If a sufficient amount of expression is detected in agricultural products or goods, then they are expected to contain a nucleotide sequence that can diagnose the presence of rvVMO858 maize transgenic object material in the agricultural products or goods. Agricultural products and commodities include, but are not limited to, corn oil, corn meal, corn meal, corn gluten, corn meal, corn starch, and any other food product that can be used as a food source for animal consumption or otherwise case as a leavening agent for dough or ingredients in cosmetic compositions for cosmetic use, etc. It is possible to develop a method of detecting nucleic acids and/or a set based on a probe or a pair of primers for detecting the nucleotide sequence of the transgenic object of corn YVMOU858, such as those proposed in ZEO IU MO: 1 or 5EO IUO MO 2, in a biological sample to detect the presence of a transgenic object OVMOU858 maize object, where the probe sequence or primer sequence is selected from the sequence proposed in ZEO IU MO: 1, 5EO I MO: 2, ZEO IU MO: 3,

ZEOIU MO: 4 and ZEOIU MO: 5.

In conclusion, the transgenic object of corn OVMO9858 according to the present invention has good resistance to the herbicide glyphosate and the herbicide glufosinate, and does not affect the yield, and the method

Zo detection can accurately and quickly detect whether a biological sample contains a DNA molecule from the OVMO858 transgenic object of corn.

The seeds of the transgenic object of maize OVMO858 were deposited in the Center of the General Microbiological Collection of Cultures of China (abbreviation of СОМСС, address: Ipbiyshe oi

Mistorioiodu, Spipese Asadetu oi bsiepse5, Mo.1 Veispep UUezi Voaid, Snpaosuapo bivinisi, Veiipo, index 100101) on December 24, 2014 in the category by name: corn (7ea tauz), deposit number of СОМСС Mo.10212. The deposit will be kept in storage for 30 years.

Finally, it should be noted that the above examples are given only to illustrate the technical solutions of the present invention and do not limit the invention. Although the present invention has been described in detail with reference to preferred examples, it will be understood by those skilled in the art that any modification or equivalent substitution can be made in the technical solutions of the present invention within the spirit and scope of the technical solutions of the present invention.

LIST OF SEQUENCES

"1105 VEI0IMO RAVEIMOMS TESNMOTOSU SVOOR CO., ITO.

VEIChT2IMS RAVEIMOMO VIOTESNMOTOSU SO., ITr. "1205 CORN PLANT OVM9858, RESISTANT TO HERBICIDE AND NUCLEOTIDE

SEQUENCE AND METHOD FOR ITS DETECTION

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Yessdayesses dssdEsdssud ssdsseEssods ddaaddddsud assdedayeda asddeEssdda 5160 adaaseEssds dESsSaaddaaa dsdassudsseyi seESsSddssudEyes dssaayedudses Esaadsesaa 5220 bdadasodeddae sdsdddayedadcc dsdadasdes dseEsdEsdvd soesd dssuss seEdasuddsaa 5280 chadsEsdds aasudssyessdu dsdssodssuyeE sussasssaye sesudayesasss dsayesdssaye 5340 chadsEyesses dESayeddudes EsdEdeEsodda aaasssedEsS asduddyeddasu ayedssasschdae 5400 daysssssud adsEyssssdud adessayedda sseydayesdd ss ddassdoddso sddaadaesda 5460 asEsESssdae asdaaddssd sseEdaassad Yedaesudeessa aasaesveduds aasaaadeve 5520 sEeaadchaesu aayesssdeEvd ssudESEevds daadayataes asasaaaseyes sooseEdaaeta 5580 sdeeaaadsaye dsaaeaaaesta asaedeaaesu saedassuesta EsSEaedaday doEsSEsrae 5640 chaesadades ssdsaaesta asaaeesaara susdasbadaa aaaasaae adsdsdsaaa 5700 bo ssaddayebaaa Sbassoosdsud sododeEdEsayes gayedeEsasta daeyssoddayess sdgddeEssya 5760 dESsSssYadesva aasssaaesa deESsadesdssud dEdadadsudye adseEdsstdu aadsddseseyes 5820

ChdEsadssadE YedadadsadeE daadaddduds seEsadayessa doaodsdsdsaaa ddsssaaaasa 5880 assadsssas sadsseEsaseE sdadaaesad sudseEsaseyesa seEsudssssad sudeEsuyeYedas o 5940 chCHEaddssbad seEssayesaa asayedsayedda Yedaydsvaad saadaaaaaa assesasts 6000 ayesaaaaaa Sudsaaaayesye EedesyaochdChdeye adseEdadsss dasaaaadeaa adaseysstach 6060 sEeyodssoyebu sagdsSue yedsssssveE daasesdayes daysdasaayess sayeddassaye 6120 chdsadseEvdseE seEddesssEeE sssEvbudYeessE dsgdsayesda deEedssss so sssssssssu 6180 dEaassasad aaddayesdsa EedEasuees ssaedsedaa ddasodudeeda addayesaddaa 6240 aadchdadasda deaddEeEssaa daayessadad adsueaasuye YessdseEsSsaye gdsssayesda 6300 sayeesassaa assdaadsaye saadssayesa assayessa asuddessayes sayessayessa 6360 bdsdbasaeeee eeEsdaadaad dssodessaba gdssoddEdds ddabassuesta dssayetaaas. 6420

EDESSSSSYABEDEd dsseEsSssESSSSa aeysassesss sESSESEEsSEeES sEdasaddas asayessayeu 6480 chaseEdsayedE addssadsss addaayessayed dsasdaaasud sayedayessYaae gdssudssad 6540 dsssssssud dasasasudaa ddssssssa sarsssduded ssuddsesars dsseESsudssyae s 66oo aadaeddsaa addasssada ddaasdayed sayeddayesd Yesssassaad ЧчЭЖЧЯЯЭЭеса 6б6vo deEаедаадаа Eсааесааес асаааесаад badedсаесте Ssадсесаве ssаааедес 6720 чаеадаааеда ассааеда асааааеда 6780 чаеаааеда. garden seEsayessaye Essasdaass aaasadaaa deEdaadaasa adaadaessaye 6840 adeEeeevsasses arsesesaa ssaaaasss sasaaaavreee ssdesssdaa 6890 "2105 6 "2115 234 "212" DNA "2135 Artificial sequence "220" "223" Sequence located inside 5KO I MO: 3, which covers the sequence of the DNA construct OVMI10006b and the sequence of the promoter rg355 "400 5 6 chchvaeavaeed yeddeEdsaaas aaaeeedasds sSsadasaasoi Saasasasa ssdsddayegas bo

From chassayesdsed dssdsssagba Saaddsdsuds sayeddadssa aadayessaaa gbadaddasse 120 aasadaasees dssdebaaada seddsdaasa dsssasasad adssssssas dasssaaysdva 180 saadaadaaa asssssddyesa asasddsdda dsasdasasud seEgdEsStase ssaa 234 "2105 17

C5 "2115" 248 "212" DNA "2135 Artificial sequence "220" "223" Sequence located inside 5BO I MO: 4, which includes the sequence of the terminator ЕМо5 and the sequence of the DNA construct ОВМ10006 "4005 7 сдесаадсае dсааааесса асадсааесда саедасааесааеа doaodeEseevae bo chayesadadeEs ssdsaayetaye asaeesasaasa sdsdasadaa asaaaaase adsdsdsaaa 120 staddayaaaa Yesayesdsoosdu soodeEdESaVayes gasdeyesasta dayesddchasss sdYadgyeddayesyessssd 180 dESSsSsSYAEEEeeva aasstaaesa deESsadsdsso desEdadadssds adsssssssssssssss sddseses 240 dEsadssa 248 "2105 8 "2115 23 "212" DNA "2135 Artificial sequence "220" "223" First primer for amplification of 5BO and MO : With "4005 8 ayedsssvdss Yedasssasvyed aad 23 "2105 19 60 "2115 25 "212" DNA

Zo

"2135 Artificial sequence "223" Second primer for 5BO I MO amplification: Z "4005 9

Egddadeada saadsuedeEsS dEdse 25 "2105 10 "2115 30 "212" DNA "2135 Artificial sequence "2205 "223" First primer for amplification of 5BO I MO: 4 "4005 10 sdeeaadsaye dsaaeaaayeta asaydeaaed 30 "2105 11 "2115 2 5 "212" DNA " 2135 Artificial sequence "2205 "223" Second primer for amplification 5BO I MO: 4 "4005 11 bEsaddasdd aaayeseEsayed dadka 25 "2105 12 "2115 26 "2125" DNA "2135 Artificial sequence "2205 "223" Primer on 5'-flanking genomic sequences "4005 12 chdsaddsadee adseESEDdaae aasasd 26 "2105 13 "2115 21 "212" DNA "2135 Artificial sequence "2205 "223" Primer located on T-DNA paired to 5BO IRR MO: 12 "4005 13 asdddschdadeEsse seEdEsaddes s 21 " 2105 14 "2115 24 "2125" DNA "2135 Artificial sequence "2205 "223" Primer on the 3'-flanking genomic sequence "4005 14 adsaeasasd dssaadstad dade 24 "2105 15 "2115 20 6o0 "2125" DNA "2135 Artificial sequence

"223" Primer located on T-DNA, paired to 5EO I MO: 14 "4005 15 dsEdssedda adsddsesesd 20 "2105 16 "2115 25 "212" DNA "2135 Artificial sequence "2205 "223" Primer 1 for detection of EROBR5 by Tadtstap "4005 16 seddaaddsu addasudesaye saatga 25 "2105 17 "2115 22 "212" DNA "2135 Artificial sequence "2205 "223" Primer 2 for detection of EROBR5 by Tadtstap "4005 17

Yedasdasayee dssdaaayesd ad 22 "2105 18 "2115 28 "212" DNA "213" Artificial sequence "2205 "223" Probe 1 for detection of ERBR5 by Tatsdtshap "4005 18 agyedsaddsda gdddsssdud sayessdbta 28 "2105 19 "2115 24 "2 12" DNA "2135 Artificial sequence "2205" 223 "Primer 3 for the detection of RAT by Takdtshap" 4005 19 Sadesdesaddsadsee Assad 24 "2105 20" 2115 27 "212" DNA "213" artificial sequence "2205" 223 "4005" for detection 20

SESaseyedsad asdeEsssaae dsaayesda 27 "2105 21 "2115 24 "212" DNA 6o0 "213" Artificial sequence "2205

"223" Probe 2 for detection of RAT by Tatsdtshap "4005 21 sadseEdavaye dadsssoudeEe ed9dEd 24 "2105 122 "2115 23 "2125 DNA "2135 Artificial sequence "220" "223" first primer for endogenous corn ubiquitin gene "4005 D 22 adsadasd ds asddsayeses de 23 "2105 23 "2115 27 "2125 DNA "2135 Artificial sequence "220" "223" second primer for endogenous maize ubiquitin gene "4005 23 sadadaadsada asgrassdddds ssegaass 27 "2105 24 "2115" 310 "2125 DNA "2135 Artificial sequence

From "2205 "223" probe for RAT for detection of hybridization by Southern blotting "4005 24 sadasssaaa assssdsss Sssasadase gaadsaaard edeEdsasaae deddayessta bo chdasssaasss EeDDChaedssta sSdEdasasuye aaasadvaseE sssaaseedeEsS saayesoaeaad 120 sdEsssssads sesssad ddes ssadsudbraad saasassads sasaasasss ssaassesad 180 saassaassa adddssayesessa sSsssdsaass ЕСЕСТадес assaаессасес ЕЕЕССЯеЕ9аЧЕЯ 240

ЕЕЕЭЧЕЧ9ЧОССЕС EDESSvaaad ЕЕЕСассдсад асдссссаае dsaasddeEsba асдгагбаесас Zoo aaassdsdds 310 "2105 25 "2115 304 "2125 DNA "2135 Artificial sequence "220" "223" probe for EROBRO for detection of hybridization by southern blotting "4005 225

Чдаесассудес ЕЕсСсдаевеесасеедсасдс ссассссдосд садсддеесс аасасдсдадас бо ссајесддасу сеЕгдсдадс daddsdeEсудс соаедчаадЧче dсЕдссддааа Сдбадассс 120 сдасдадсс сассдсад сддсадссуд gddсодсаее dссдаааессд adsddssdsse 180 saddsssad daddssdssa Ssdssdasds sayesdayadee ssaddeEdesd sseEsssetas 240 chchvaedsddds dsssayesdses edsayeddsesso sSdsssdoabaess dahsdasdsess sSsdassesssa Zoo chaad 304 "2105 26 "2115 19 "2125 DNA "2135 Artificial sequence bo "2205

"223" primer located on T-DNA, in the same direction as 5BO ID MO: "4005 26 aadsdedeEsdd Yedsessass 19 "2105 hЖ27 "2115 20 "2125 DNA "2135 Artificial sequence "2205 "223" primer located on T-DNA, in the opposite direction to 5EO Ii

MO: 13 "4005 227 ssasssasda ddadsayesdChe 20 "2105 28 "2115 20 "2125 DNA "2135 Artificial sequence "2205 "223" primer located on T-DNA, in the opposite direction to 5EO Ii

MO: 13 "4005 28

Yedasdeaadd dahedasdsas 20 "2105 29 "2115 20 "2125" DNA "2135 Artificial sequence "2205 "223" primer located on T-DNA, in the same direction as 5BO ID MO: 15

З5 "4005 229 assay Esadeedss4d9dd 20 "2105 30 "2115 24 "2125 DNA "2135 Artificial sequence "2205 "223" primer located on T-DNA, in the opposite direction to 5EO Ii

MO: 15 "4005 30 chdsdddaseEseE aassagaaaaa asss 24 "2105 31 "2115 20 "2125 DNA "2135 Artificial sequence "2205 "223" primer located on T-DNA, in the opposite direction to 5EO Ii

MO: 15 "4005 31 sdsaadassu dsaasaddaye 20 60

Claims (18)

FORMULA OF THE INVENTION 1. A nucleic acid molecule for detecting the presence of ZEO IU MO: 5 or its complementary sequence in a sample, where the nucleic acid sequence contains ZEO IUO MO: 1 or its complementary sequence and/or ZEO IU MO: 2 or its complementary sequence. 2. Nucleic acid molecule according to claim 1, where the nucleic acid sequence contains ZEO IU MO: C or its complementary sequence and/or 5EO IO MO: 4 or its complementary sequence. Z. Nucleic acid molecule according to claim 2, where the nucleic acid sequence contains ХЭО ИЯ МО: 5 or its complementary sequence. 4. A method of detecting the presence of ZEO IU MO: 5 or its complementary sequence in a sample, which includes: contact of the sample for detection with at least two primers for amplification of the product of interest in the nucleic acid amplification reaction; conducting a nucleic acid amplification reaction; and detecting the presence of the amplification product of interest; where the amplification product of interest contains 5EO IUO MO: 1 or its complementary sequence and/or 5EO IUO MO: 2 or its complementary sequence. 5. The method according to claim 4, where the amplification product of interest additionally contains ZEO IU MO: 6 or its complementary sequence and/or 5EO IO MO: 7 or its complementary sequence. 6. The method according to claim 4 or 5, where at least one of the primers contains the nucleic acid sequence according to any of claims 1-3 or its complementary sequence. 7. The method according to claim 6, where the primers include a first primer selected from 5EO IU MO: 8 and 5EO IU MO: 10, and a second primer selected from ZEO IUO MO: 9 and ZEO IU MO: 11. 8. Method for detecting the presence of FEO IU MO: 5 or its complementary sequence in a sample, which includes: contact of the sample for detection with a probe containing ZEO IU MO: 1 or its complementary sequence and/or 5EO IUO MO: 2 or its complementary sequence ; For hybridization of the sample for detection with the probe under strict hybridization conditions; and detecting hybridization of the detection sample with the probe. 9. The method according to claim 8, where the probe additionally contains or has 5EO IU MO: 6 or its complementary sequence and/or 5EO IUO MO: 7 or its complementary sequence. 10. The method according to any one of claims 8 or 9, wherein the probe is a nucleic acid marker molecule, and a marker-crossover analysis is used to determine whether glyphosate resistance and/or glufosinate resistance is genetically linked to the marker molecule nucleic acid. 11. The method according to any one of claims 8-10, where at least one of the probes is labeled with a fluorescent group. 12. A kit for detecting DNA, including at least one DNA molecule, where the DNA molecule contains ZEO IU MO: 1 or its complementary sequence and/or ZEBO IO MO: 2 or its complementary sequence and is suitable as a DNA primer or a DNA probe, specific to ZEOIYUO MO: 5. 13. The kit for detecting DNA according to claim 12, where the DNA molecule additionally contains 5EO IU MO: 6 or its complementary sequence and/or ZEO IU MO: 7 or its complementary sequence. 14. A plant cell or part for obtaining a transgenic corn plant containing the nucleic acid sequence ZEO IUO MO: 1, the nucleic acid sequence of nucleotides 1157-5744 5EBEO IO MO: 5 and 5EBO I MO: 2, arranged sequentially, or the nucleic acid sequence 5EO IU MO: 5. 15. A method of obtaining a transgenic corn plant that has resistance to the herbicide glyphosate and/or the herbicide glufosinate, which includes: introducing the nucleic acid sequence represented by 1157-5744 5EBEO IJOO MO: 5 into the genome of a corn plant in such a way that the genome of the corn plant contains nucleic acid sequence ZEO IU MO: 1, nucleic acid sequences from nucleotides 1157-5744 5EO IU MO: 5 and ZEO IU MO: 2, located sequentially, or nucleic acid sequence 5EO IU MO: 5; selection of corn plants resistant to the herbicide glyphosate and/or the herbicide glufosinate. 16. A method of cultivating a corn plant that has resistance to the herbicide glyphosate and/or the herbicide glufosinate, which includes: planting at least one seed of corn that contains in its genome the sequence of 60 nucleic acids of the specified area, where the nucleic acid sequence of the specified area contains ZEO IU MO : 1, nucleic acid sequence from nucleotides 1157-5744 5EO IO MO: 5 and ZEO IO MO: 2, located in sequence, or nucleic acid sequence 5EO IO MO: 5; growing corn seed to corn plant; spraying the corn plant with an effective dose of the herbicide glyphosate and/or the herbicide glufosinate, and harvesting the corn plant with reduced damage to the plant compared to other plants that do not contain the nucleic acid sequence in the specified region. 17. A method of controlling glyphosate-resistant weeds in a field with glyphosate-resistant plants, which comprises: applying a herbicide containing an effective dose of glufosinate to a field planted with at least one transgenic glyphosate-resistant corn plant, wherein the transgenic maize plant resistant to glyphosate contains in its genome ZEO IU MO: 1, the nucleic acid sequence of nucleotides 1157-5744 5EO IU MO: 5 and 5EO IUO MO: 2, arranged in sequence, or the transgenic maize plant contains in its genome ZEO IU MO: 5, and a transgenic corn plant resistant to glyphosate is also resistant to the herbicide glufosinate. 18. A method of slowing the development of resistance in insects, which includes planting at least one transgenic corn plant with resistance to glyphosate and/or glufosinate in a field where an insect-resistant corn plant is planted, where the transgenic corn plant with resistance to glyphosate and/or glufosinate contains in its genome ZEO IUO MO: 1, a nucleic acid sequence of nucleotides 1157-5744 5BO IO MO: 5 1 5BO IO MO: 2, located in sequence, or the transgenic corn plant contains in its genome ZEO IUO MO: 5. YAEK NU MEK I "my my - ZVO Mo: a ZHEO SCHE MO: guilty shen wine ZVO MO: 7 BEOSHCMO Ann MO vania i - 80: t 7 | : i Y x Z sSate Yevloyae B'yertya bevole iv ! . | EV and ! | : | I. 0 Che sRAT: and sir what! immediately rel FIG. 1 Зб в в "er" DOD тя ихо5 о5 ; her TU vrastrI her PUE. VAK inniv p Di roza ih V M still what B shchi ekv nn t, rK35 sh ZA