CN116656870B - Transgenic soybean event LP086-3 and detection method thereof - Google Patents

Transgenic soybean event LP086-3 and detection method thereof Download PDF

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CN116656870B
CN116656870B CN202310912451.1A CN202310912451A CN116656870B CN 116656870 B CN116656870 B CN 116656870B CN 202310912451 A CN202310912451 A CN 202310912451A CN 116656870 B CN116656870 B CN 116656870B
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soybean
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CN116656870A (en
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贾志伟
吕玉平
梅方明
赵丽媛
邸萌亮
李涛
李程成
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Longping Biotechnology Hainan Co ltd
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    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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Abstract

The invention provides a nucleic acid sequence comprising one or more selected from the sequences SEQ ID NO. 1-7 or the complement thereof, said nucleic acid sequence being derived from transgenic soybean event LP086-3, a representative sample of seed comprising said event having been deposited under accession number CCTCC NO. P202327. The transgenic soybean event LP086-3 of the present invention has the following advantages: the economic loss caused by lepidopteran pests is avoided; soybean crops that are tolerant to the common commercial herbicide glyphosate; the soybean yield is not reduced; enhancing breeding efficiency, enabling the use of molecular markers to track transgene inserts in the breeding populations and their offspring. Meanwhile, the detection method provided by the invention can rapidly, accurately and stably identify the existence of the plant material derived from the transgenic soybean event LP 086-3.

Description

Transgenic soybean event LP086-3 and detection method thereof
Technical Field
The invention belongs to the technical field of molecular biology, relates to a detection method of transgenic plants and products thereof, and in particular relates to transgenic soybean event LP086-3 which is resistant to insects and resistant to glyphosate herbicide application, and a nucleic acid sequence and a method for detecting the transgenic soybean LP 086-3.
Background
Soybeans (Glycine max (linn.) merr.) are important food and oil crops in many parts of the world. Biotechnology has been applied to soybeans to improve their agronomic traits and quality. Insect resistance is an important agronomic trait in soybean production, particularly resistance to lepidopteran insects (e.g., soybean borer, spodoptera frugiperda, asparagus caterpillar, prodenia litura, cotton bollworm, etc.). Resistance of soybean to lepidopteran insects can be obtained by expressing a lepidopteran insect resistance gene in a soybean plant by a transgenic method. Another important agronomic trait is herbicide tolerance, particularly glyphosate tolerance. Tolerance of soybeans to glyphosate herbicide can be achieved by transgenic approaches to express glyphosate herbicide tolerance genes (e.g., epsps) in soybean plants.
In addition to the functional genes (epsps gene and pat gene) themselves, the choice of regulatory elements and their sequential arrangement are crucial for obtaining good transformation events and their technical effects are unpredictable. It is also known that the expression of foreign genes in plants is affected by their insertion into the soybean genome, possibly due to the proximity of chromatin structures (e.g., heterochromatin) or transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events to make it possible to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the expression level of the introduced gene may vary greatly between events; there may also be differences in the spatial or temporal pattern of expression, such as differences in the relative expression of transgenes between different plant tissues, which differences may be manifested in actual expression patterns that are inconsistent with the expression patterns expected for the transcriptional regulatory elements in the introduced gene construct. Thus, it is often desirable to generate hundreds or thousands of different events and screen those events for a single event having transgene expression levels and patterns that are expected for commercialization purposes. Such transformation events have excellent lepidopteran pest (e.g., soybean borer, spodoptera frugiperda, asparagus caterpillar, prodenia litura, cotton bollworm, etc.) and glyphosate herbicide resistance without affecting soybean yield, and conventional breeding methods can be used to backcross transgenic traits into other genetic backgrounds by crossing. The progeny produced by this crossing maintains the transgene expression characteristics and trait performance of the original transformant. The application of the strategy mode can ensure reliable gene expression in a plurality of varieties, has stable lepidoptera pests (such as soybean borer, spodoptera frugiperda, beet armyworm, prodenia litura, cotton bollworm and the like) and glyphosate herbicide resistance, prevents the varieties from being harmful to main lepidoptera pests, has broad-spectrum weed control capability, and can be well adapted to the growth conditions of places.
It would be beneficial to be able to detect the presence of a particular event to determine whether the progeny of a sexual cross contain a gene of interest. In addition, methods of detecting specific events will also help to comply with relevant regulations, such as the need for formal approval and marking of foods derived from recombinant crops prior to their being put on the market. It is possible to detect the presence of the transgene by any well known polynucleotide detection method, 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, and the like. Thus, unless the sequence of chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, such a method as described above cannot be used to distinguish between different events, particularly those generated with the same DNA construct. Therefore, it is common today to identify a transgene specific event by PCR using a pair of primers spanning the junction of the inserted T-DNA and flanking DNA, specifically a first primer comprising the flanking sequence and a second primer comprising the inserted sequence.
Disclosure of Invention
The invention aims to provide a transgenic soybean event LP086-3, a nucleic acid sequence for detecting soybean plant LP086-3 event and a detection method thereof, which can accurately and rapidly identify whether a biological sample contains DNA molecules of specific transgenic soybean event LP 086-3.
To achieve the above object, the present invention provides a nucleic acid sequence comprising one or more selected from the sequences SEQ ID NO 1-7 (i.e., SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7) or the complement thereof. In some embodiments, the nucleic acid sequence is derived from a plant, seed, or cell comprising soybean event LP086-3, a representative sample of seed comprising the event having been deposited at the chinese typical culture collection (CCTCC, address: eight of the kangaroo regions of the marchan district of the hubei province, university of marchan, accession No. 430072) under accession number cctccc No. P202327 at 2023, classification nomenclature: soybean seed LP086-3 (Glycine max l. LP 086-3). In some embodiments, the nucleic acid sequence is an amplicon diagnostic for the presence of soybean event LP 086-3.
In some embodiments of the invention, the invention provides a nucleic acid sequence comprising at least 11 consecutive nucleotides of SEQ ID NO. 3 or a complement thereof and/or at least 11 consecutive nucleotides of SEQ ID NO. 4 or a complement thereof. In some embodiments, the nucleic acid sequence comprises SEQ ID NO. 1 or a complement thereof, and/or SEQ ID NO. 2 or a complement thereof. In some embodiments, the nucleic acid sequence comprises SEQ ID NO. 3 or a complement thereof, and/or SEQ ID NO. 4 or a complement thereof. In some embodiments, the nucleic acid sequence comprises SEQ ID NO. 5 or a complement thereof.
The SEQ ID NO. 1 or the complementary sequence thereof is a sequence with the length of 56 nucleotides, which is positioned near the insertion junction at the 5 '-end of the insertion sequence in the transgenic soybean event LP086-3, the SEQ ID NO. 1 or the complementary sequence thereof spans the flanking genomic DNA sequence of the soybean insertion site and the DNA sequence at the 5' -end of the insertion sequence, and the existence of the transgenic soybean event LP086-3 can be identified by comprising the SEQ ID NO. 1 or the complementary sequence thereof. The SEQ ID NO. 2 or the complementary sequence thereof is a sequence with the length of 49 nucleotides, which is positioned near the insertion junction at the 3 '-end of the insertion sequence in the transgenic soybean event LP086-3, the SEQ ID NO. 2 or the complementary sequence thereof spans the DNA sequence at the 3' -end of the insertion sequence and the flanking genomic DNA sequence of the soybean insertion site, and the existence of the transgenic soybean event LP086-3 can be identified by comprising the SEQ ID NO. 2 or the complementary sequence thereof.
The nucleic acid sequences provided by the invention may be at least 11 or more contiguous polynucleotides (first nucleic acid sequences) of any portion of the transgene insert sequence in SEQ ID NO. 3 or its complement, or at least 11 or more contiguous polynucleotides (second nucleic acid sequences) of any portion of the 5' flanking soybean genomic DNA region in SEQ ID NO. 3 or its complement. The nucleic acid sequence may further be homologous or complementary to a portion of the SEQ ID NO. 3 comprising the complete SEQ ID NO. 1. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences comprise a pair of DNA primers in a DNA amplification method that produces an amplification product. The presence of transgenic soybean event LP086-3 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO. 1. It is well known to those skilled in the art that the first and second nucleic acid sequences need not consist of only DNA, but may include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA, or other nucleotides or analogs thereof that do not serve as templates for one or more polymerases. Furthermore, the probes or primers described in the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which may be selected from the nucleotides set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. When selected from the group consisting of the nucleotides set forth in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, the probes and primers may be about 17 to 50 or more consecutive nucleotides in length. The SEQ ID NO. 3 or its complement is a 1154 nucleotide long sequence located near the 5 'end of the insertion junction in the transgenic soybean event LP086-3, and the SEQ ID NO. 3 or its complement consists of 560 nucleotide soybean flanking genomic DNA sequences (nucleotides 1-560 of SEQ ID NO. 3), 376 nucleotide pLP086 construct DNA sequences (nucleotides 595-970 of SEQ ID NO. 3) and 184 nucleotide 3' end DNA sequences of the pAtUbi10 promoter (nucleotides 971-1154 of SEQ ID NO. 3), which are included to identify the presence of the transgenic soybean event LP 086-3.
The nucleic acid sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid sequence) of any portion of the transgene insert sequence in the SEQ ID NO. 4 or its complement, or at least 11 or more contiguous nucleotides (fourth nucleic acid sequence) of any portion of the 3' flanking soybean genomic DNA region in the SEQ ID NO. 4 or its complement. The nucleic acid sequence may further be homologous or complementary to a portion of the SEQ ID NO. 4 comprising the complete SEQ ID NO. 2. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, the method of amplifying DNA to produce an amplified product includes a pair of DNA primers. The presence of transgenic soybean event LP086-3 or its progeny can be diagnosed when the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO. 2. The sequence of SEQ ID NO. 4 or its complement is a sequence of 880 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in transgenic soybean event LP086-3, the SEQ ID NO. 4 or its complement consists of 314 nucleotides of the tPsE9 terminator partial sequence (nucleotides 1-314 of SEQ ID NO. 4), 207 nucleotides of the pLP086 construct DNA sequence (nucleotides 315-521 of SEQ ID NO. 4) and 330 nucleotides of the soybean integration site flanking genomic DNA sequence (nucleotides 551-880 of SEQ ID NO. 4), and the inclusion of the SEQ ID NO. 4 or its complement can be identified as the presence of transgenic soybean event LP 086-3.
The SEQ ID NO. 5 or its complement is a sequence of 17158 nucleotides in length characterizing transgenic soybean event LP086-3, which specifically contains the genome and genetic elements as shown in Table 1. The presence of transgenic soybean event LP086-3 can be identified by inclusion of said SEQ ID NO. 5 or its complement.
Table 1, genome and genetic element contained in SEQ ID NO. 5
The nucleic acid sequence or the complement thereof may be used in a DNA amplification method to produce an amplification product, the presence of the transgenic soybean event LP086-3 or its progeny in a biological sample being diagnosed by detection of the amplification product; the nucleic acid sequence or the complement thereof may be used in a nucleotide assay to detect the presence of transgenic soybean event LP086-3 or a progeny thereof in a biological sample.
The present invention provides a DNA primer pair comprising a first primer and a second primer, wherein each of the first primer and the second primer comprises a fragment of SEQ ID No. 5 or a complement thereof and when used in an amplification reaction with DNA comprising soybean event LP086-3, produces an amplification product that detects soybean event LP086-3 in a sample.
In some embodiments, the first primer is selected from the group consisting of SEQ ID NO. 1 or a complement thereof, SEQ ID NO. 8 or SEQ ID NO. 10; the second primer is selected from SEQ ID NO. 2 or a complementary sequence thereof, SEQ ID NO. 11 or SEQ ID NO. 14.
In some embodiments of the invention, the amplification product comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement.
Further, the amplification product comprises consecutive nucleotides 1 to 44 or 45 to 56 in SEQ ID NO. 1 or its complement, or consecutive nucleotides 1 to 10 or 11 to 49 in SEQ ID NO. 2 or its complement.
Still further, the amplification product comprises SEQ ID NO. 1 or its complement, SEQ ID NO. 2 or its complement, SEQ ID NO. 6 or its complement, or SEQ ID NO. 7 or its complement.
In the above technical scheme, the primer comprises at least one of the nucleic acid sequences. Specifically, the primer comprises a first primer and a second primer, wherein the first primer is selected from SEQ ID NO. 1 or a complementary sequence thereof, SEQ ID NO. 8 or SEQ ID NO. 12, and the second primer is selected from SEQ ID NO. 9 or SEQ ID NO. 13; or the first primer is selected from SEQ ID NO. 2 or a complementary sequence thereof, SEQ ID NO. 10 or SEQ ID NO. 15, and the second primer is selected from SEQ ID NO. 11 or SEQ ID NO. 14.
The present invention also provides a DNA probe comprising a fragment of SEQ ID NO. 5 or a complementary sequence thereof, which hybridizes under stringent hybridization conditions to a DNA molecule comprising a nucleic acid sequence selected from SEQ ID NO. 1-7 or a complementary sequence thereof and does not hybridize under stringent hybridization conditions to a DNA molecule not comprising a nucleic acid sequence selected from SEQ ID NO. 1-7 or a complementary sequence thereof.
In some embodiments, the DNA probe comprises a sequence selected from the group consisting of SEQ ID NO. 1 or its complement, SEQ ID NO. 2 or its complement, SEQ ID NO. 6 or its complement, and SEQ ID NO. 7 or its complement.
In some embodiments, the DNA probe is labeled with a fluorescent group.
In some embodiments, the probe comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement; further, the probe comprises continuous nucleotides at positions 1-44 or 45-56 in SEQ ID NO. 1 or the complementary sequence thereof, or continuous nucleotides at positions 1-10 or 11-49 in SEQ ID NO. 2 or the complementary sequence thereof.
The present invention also provides a marker nucleic acid molecule comprising a fragment of SEQ ID NO. 5 or a complement thereof, which hybridizes under stringent hybridization conditions with a DNA molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO. 1-7 or a complement thereof and does not hybridize under stringent hybridization conditions with a DNA molecule not comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO. 1-7 or a complement thereof.
In some embodiments, the marker nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO. 1 or its complement, SEQ ID NO. 2 or its complement, SEQ ID NO. 6 or its complement, and SEQ ID NO. 7 or its complement.
In one embodiment, the marker nucleic acid molecule comprises at least 11 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 11 consecutive nucleotides of SEQ ID NO. 4 or its complement;
in some embodiments, the marker nucleic acid molecule comprises consecutive nucleotides 1-44 or 45-56 of SEQ ID NO. 1 or its complement, or consecutive nucleotides 1-10 or 11-49 of SEQ ID NO. 2 or its complement.
Further, the present invention provides a method for detecting the presence of DNA comprising transgenic soybean event LP086-3 in a sample, comprising:
(1) Contacting a sample to be detected with the pair of DNA primers in a nucleic acid amplification reaction;
(2) Performing a nucleic acid amplification reaction;
(3) Detecting the presence of an amplification product;
the amplification product comprises a nucleic acid sequence selected from the sequences SEQ ID NO. 1-7 or the complement thereof, i.e.is indicative of the presence of DNA comprising transgenic soybean event LP086-3 in the test sample.
The invention also provides a method of detecting the presence of DNA comprising transgenic soybean event LP086-3 in a sample, comprising:
(1) Contacting a sample to be detected with said DNA probe, and/or said marker nucleic acid molecule;
(2) Hybridizing the sample to be detected with the probe and/or the marker nucleic acid molecule under stringent hybridization conditions;
(3) Detecting hybridization of the sample to be detected with the probe and/or the marker nucleic acid molecule.
The stringent conditions may be hybridization in 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65℃and then washing the membrane 1 time with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS, respectively.
Wherein hybridization of the sample to be tested and the marker nucleic acid molecule is detected, and further by marker assisted breeding analysis to determine that insect resistance and/or herbicide tolerance is genetically linked to the marker nucleic acid molecule.
The invention also provides a DNA detection kit, comprising: a DNA primer pair that produces an amplicon diagnostic for transgenic soybean event LP086-3, a probe specific for SEQ ID NOs 1-7 or a marker nucleic acid molecule specific for SEQ ID NOs 1-7. Specifically, the detection kit comprises the probe, the primer pair or the marker nucleic acid molecule.
In some embodiments, the invention provides a DNA detection kit comprising at least one DNA molecule comprising at least 11 consecutive nucleotides of the homologous sequence of SEQ ID NO. 3 or the complement thereof, or at least 11 consecutive nucleotides of the homologous sequence of SEQ ID NO. 4 or the complement thereof, which can be used as a DNA primer or probe specific for transgenic soybean event LP086-3 or a progeny thereof.
Further, the DNA molecule comprises continuous nucleotides at positions 1-44 or 45-56 in SEQ ID NO. 1 or the complementary sequence thereof, or continuous nucleotides at positions 1-10 or 11-49 in SEQ ID NO. 2 or the complementary sequence thereof.
Still further, the DNA molecule comprises a homologous sequence of SEQ ID NO. 1 or a complement thereof, a homologous sequence of SEQ ID NO. 2 or a complement thereof, a homologous sequence of SEQ ID NO. 6 or a complement thereof, or a homologous sequence of SEQ ID NO. 7 or a complement thereof. To achieve the above object, the present invention also provides a plant cell comprising nucleic acid sequences encoding insect-resistant Cry1Ab, cry2Ab and Cry1Fa proteins, a nucleic acid sequence encoding a glyphosate herbicide tolerance EPSPS protein and a nucleic acid sequence of a specific region comprising the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO: 7.
The sequences provided by the present invention include the sequences listed in table 2 below:
TABLE 2 related sequences of the invention
The present invention also provides a method of protecting a soybean plant from insect infestation comprising providing at least one transgenic soybean plant cell in the diet of a target insect, said transgenic soybean plant genome comprising in sequence SEQ ID NO. 1, SEQ ID NO. 5, nucleic acid sequence from position 606 to 16789, and SEQ ID NO. 2; or the genome of the transgenic soybean plant comprises a sequence shown in SEQ ID NO. 5; target insects that ingest the transgenic soybean plant cells are inhibited from further ingest the soybean plant.
The present invention also provides a method of protecting a soybean plant from injury caused by a herbicide, planting at least one transgenic soybean plant comprising in sequence SEQ ID NO. 1, SEQ ID NO. 5, nucleic acid sequence from position 606 to 16789, and SEQ ID NO. 2 in the genome of the transgenic soybean plant; or the genome of the transgenic soybean plant comprises a sequence shown as SEQ ID NO. 5. In some embodiments, the method comprises applying an effective dose of glyphosate herbicide to a field in which at least one transgenic soybean plant is grown, the transgenic soybean plant being transgenic soybean event LP086-3.
The present invention also provides a method of controlling weeds in a field in which soybean plants are grown, comprising applying to the field in which at least one transgenic soybean plant is grown an effective dose of a glyphosate herbicide, said transgenic soybean plant comprising in genome in sequence SEQ ID NO. 1, the nucleic acid sequence of SEQ ID NO. 5 at positions 606-16789, and SEQ ID NO. 2; or the genome of the transgenic soybean plant comprises a sequence shown as SEQ ID NO. 5.
The present invention also provides a method of culturing a soybean plant resistant to insects comprising: planting at least one soybean seed, wherein the genome of the soybean seed sequentially comprises a nucleic acid sequence of SEQ ID NO. 1, 606-16789 bits of SEQ ID NO. 5 and SEQ ID NO. 2; or the genome of the soybean seed comprises a sequence shown as SEQ ID NO. 5;
Growing the soybean seeds into soybean plants;
the soybean plants are affected with a target insect, and plants are harvested having reduced plant damage as compared to other plants not the soybean seeds.
In some embodiments, the invention provides a method of growing a soybean plant that is resistant to insects and tolerant to glyphosate herbicide comprising:
planting at least one soybean seed, wherein the genome of the soybean seed sequentially comprises a nucleic acid sequence of SEQ ID NO. 1, 606-16789 bits of SEQ ID NO. 5 and SEQ ID NO. 2; or the genome of the soybean seed comprises a sequence shown as SEQ ID NO. 5;
growing the soybean seeds into soybean plants;
the soybean plants are affected with a target insect, and the soybean plants are sprayed with an effective dose of a glyphosate herbicide, and plants are harvested having reduced plant damage compared to other plants not the soybean seeds, the plants having reduced plant damage also being resistant to feeding damage by the insect.
In some embodiments, the invention also provides a method of producing a soybean plant that is resistant to insects, comprising introducing into the genome of said soybean plant a transgenic soybean event LP086-3, selecting a soybean plant that has reduced plant damage to insect ingestion. In some embodiments, the method comprises: sexual crossing a transgenic soybean event LP086-3 having resistance to an insect with a second parent soybean plant lacking insect resistance, thereby producing a plurality of progeny plants; attack the progeny plant with a target insect; selecting said progeny plants having reduced plant damage compared to other plants not having transgenic soybean event LP 086-3.
In some embodiments, the invention also provides a method of producing a soybean plant that is tolerant to a glyphosate herbicide comprising introducing into the genome of the soybean plant transgenic soybean event LP086-3 and selecting a soybean plant that is tolerant to glyphosate. In some embodiments, the method comprises: sexual crossing a transgenic soybean event LP086-3 having tolerance to a glyphosate herbicide with a second parent soybean plant lacking glyphosate tolerance, thereby producing a plurality of progeny plants; treating said progeny plants with a glyphosate herbicide; selecting said progeny plants that are tolerant to glyphosate.
In some embodiments, the invention also provides a method of producing a soybean plant that is resistant to insects and tolerant to glyphosate herbicide application, comprising: transgenic soybean event LP086-3 was introduced into the genome of the soybean plant, and a soybean plant tolerant to glyphosate and insect-resistant was selected. In some embodiments, the methods comprise sexually crossing a glyphosate tolerant and insect resistant transgenic soybean event LP086-3 first parent soybean plant with a second parent soybean plant lacking glyphosate tolerance and/or insect resistance, thereby producing a plurality of progeny plants; treating said progeny plants with glyphosate; the progeny plants that are tolerant to glyphosate are also selected to be resistant to insect feeding damage.
The present invention also provides a composition that results from transgenic soybean event LP086-3, which is soybean meal, soybean oil, soybean protein, soybean meal, okara, and the like. In some embodiments, the composition may be a soy flour, soy oil, soy protein, soy product, meal, feed, or industrial product or commodity. If sufficient expression is detected in the composition, the composition is expected to contain a nucleic acid sequence capable of diagnosing the presence of transgenic soybean event LP086-3 material in the composition. In particular, the compositions include, but are not limited to, soy flour, soy oil, soy protein, soy products, okara, any other food product that is to be consumed by an animal as a food source, or otherwise used for food industry purposes as an ingredient of soy oil or stearic acid, and the like.
The probe or primer pair-based detection methods and/or kits of the invention can be employed to detect a transgenic soybean event LP086-3 nucleic acid sequence, such as shown in SEQ ID NO. 1 or SEQ ID NO. 2, in a biological sample, wherein the probe sequence or primer sequence is selected from the sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, to diagnose the presence of the transgenic soybean event LP 086-3.
In conclusion, the transgenic soybean event LP086-3 has the dual characteristics of insect resistance and herbicide resistance, and has the following advantages: 1) Avoiding economic losses due to lepidopteran pests (e.g., cotton bollworms, spodoptera frugiperda, soybean borers, prodenia litura, asparagus caterpillar, etc., the main pests in soybean planting areas); 2) The ability to apply glyphosate-containing agricultural herbicides to soybean crops for broad spectrum weed control; 3) The soybean yield is not reduced. Specifically, the event LP086-3 of the invention has high resistance to target pests, can lead the death rate of the pests to be up to 100 percent, and protects plants to lead the pest rate to be as low as 0 percent; the herbicide composition has high tolerance to glyphosate herbicide, can safely tolerate the glyphosate spraying with the dosage of 4 times, and protects plants to ensure that the damage rate is as low as 0%; and the plant containing the event has excellent agronomic performance, and the yield percentage can reach as high as 107 percent. Furthermore, genes encoding insect resistance and glyphosate tolerance traits are linked on the same DNA segment and are present at a single locus in the transgenic soybean event LP086-3 genome, which increases breeding efficiency and enables molecular markers to be used to track transgene inserts in the breeding populations and their progeny. Meanwhile, the primer or probe sequence provided in the detection method can generate an amplification product identified as the transgenic soybean event LP086-3 or the progeny thereof, and can rapidly, accurately and stably identify the existence of plant materials derived from the transgenic soybean event LP 086-3.
Terminology
The following definitions and methods may better define the present invention and instruct those of ordinary skill in the art to practice the present invention, and unless otherwise indicated, terms are understood according to their conventional usage by those of ordinary skill in the art.
The soybean (Glycine max) and includes all plant varieties that can mate with soybeans, including wild soybean varieties.
The term "comprising" means "including but not limited to. The "processed product" refers to a product obtained by processing a raw material such as a plant or a seed, for example, a composition.
The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps (plant cones), and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. It is to be understood that parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, calli, embryos and flowers, stems, fruits, leaves and roots, which are derived from transgenic plants or their progeny which have been previously transformed with the DNA molecules of the present invention and thus at least partially consist of the transgenic cells.
The term "gene" refers to a nucleic acid fragment that expresses a particular protein, including regulatory sequences preceding (5 'non-coding sequences) and regulatory sequences following (3' non-coding sequences) the coding sequences. "native gene" refers to a gene that is found naturally to have its own regulatory sequences. By "chimeric gene" is meant any gene that is not a native gene, comprising regulatory and coding sequences found in a non-native manner. "endogenous gene" refers to a native gene that is located in its natural location in the genome of an organism. "exogenous gene" is a foreign gene that is present in the genome of an organism and that is not originally present, and also refers to a gene that has been introduced into a recipient cell by a transgenic procedure. The exogenous gene may comprise a native gene or chimeric gene inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted may be referred to as an "insertion site" or "target site".
"flanking DNA" may comprise genomic or foreign (heterologous) DNA introduced by a transformation process, such as fragments associated with a transformation event, naturally occurring in an organism such as a plant. Thus, flanking DNA may include a combination of native and foreign 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 that is immediately upstream or downstream of and adjacent to the initial exogenous inserted DNA molecule. When this flanking region is located downstream, it may also be referred to as a "left border flanking" or a "3 'genomic border region" or a "genomic 3' border sequence", etc. When this flanking region is located upstream, it may also be referred to as a "right-hand border flanking" or a "5 'genomic border region" or a "genomic 5' border sequence", etc.
Transformation procedures that cause random integration of the foreign DNA will result in transformants that contain different flanking regions that each transformant specifically contains. When recombinant DNA is introduced into plants by conventional hybridization, its flanking regions are generally not altered. Transformants will also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of heterologous DNA. "ligation" is the point at which two specific DNA fragments are ligated. For example, the junction exists where the insert DNA joins the flanking DNA. The junction point is also present in transformed organisms, where the two DNA fragments are joined together in a manner that modifies what is found in the native organism. "adapter DNA" refers to DNA that contains an adapter.
The present invention provides transgenic soybean events, termed LP086-3, and their progeny, the transgenic soybean events LP086-3 being soybean plants LP086-3, including plants and seeds of transgenic soybean events LP086-3 and plant cells thereof or regenerable parts thereof, the plant parts of transgenic soybean events LP086-3 including, but not limited to, cells, pollen, ovules, flowers, buds, roots, stems, inflorescences, leaves and products from soybean plants LP086-3, such as soybean meal, soybean flour, soybean oil, soybean milk, soybean spikes, soybean starch and biomass left in the field of soybean crops.
The transgenic soybean event LP086-3 of the invention comprises a DNA construct that, when expressed in a plant cell, confers resistance to insects and tolerance to glyphosate herbicide to the transgenic soybean event LP 086-3.
In some embodiments of the invention, the DNA construct comprises four expression cassettes in tandem, the first expression cassette comprising a suitable promoter for expression in a plant operably linked to a nucleic acid sequence of a bacillus thuringiensis insect-resistant Cry1Fa protein (Cry 1 Fa) having lepidopteran insect resistance and a suitable polyadenylation signal sequence; the second expression cassette consists of a nucleic acid sequence comprising a suitable promoter for expression in plants operably linked to an insect-resistant Cry2Aa protein (Cry 2 Aa) of bacillus thuringiensis, said Cry2Aa having lepidopteran insect resistance, and a suitable polyadenylation signal sequence; the third expression cassette comprises a suitable promoter for expression in plants operably linked to a nucleic acid sequence of a Cry1Ab protein that is predominantly resistant to lepidopteran insects and a suitable polyadenylation signal sequence. The fourth expression cassette comprises a suitable promoter for expression in plants operably linked to a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and a suitable polyadenylation signal sequence, the nucleic acid sequence of which EPSPS protein is tolerant to glyphosate herbicide. Further, the promoter may be a suitable promoter isolated from plants, including constitutive, inducible, and/or tissue-specific promoters, including, but not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort Mosaic Virus (FMV) 35S promoter, the Ubiquitin protein (Ubiquitin) promoter, the Actin (action) promoter, the agrobacterium (Agrobacterium tumefaciens) nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter, the night yellow leaf curly virus (cestron) promoter, the tuber storage protein (Patatin) promoter, the ribulose-1, 5-bisphosphate carboxylase/oxygenase (rusco) promoter, the Glutathione S Transferase (GST) promoter, the E9 promoter, the GOS promoter, the alcA/alcR promoter, the agrobacterium (Agrobacterium rhizogenes) roller promoter, and the arabidopsis (Arabidopsis thaliana) promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence for functioning in plants, including, but not limited to, polyadenylation signal sequences derived from the Agrobacterium tumefaciens (Agrobacterium tumefaciens) nopaline synthase (NOS) gene, from the cauliflower mosaic virus (CaMV) 35S terminator, from the protease inhibitor II (PIN II) gene, and from the alpha-tubulin (alpha-tubulin) gene.
In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal peptide/transit peptide nucleic acid coding sequences. The enhancer may enhance the expression level of a gene, including, but not limited to, tobacco Etch Virus (TEV) translational activator, caMV35S enhancer, and FMV35S enhancer. The signal peptide/transit peptide can direct the transport of the Cry1Ab protein and/or EPSPS protein to a particular organelle or compartment outside or inside the cell, for example, targeting to the chloroplast using a sequence encoding a chloroplast transit peptide, or targeting to the endoplasmic reticulum using a 'KDEL' retention sequence.
The Cry1Ab, cry2Ab, and Cry1Fa genes may be isolated from bacillus thuringiensis (Bacillus thuringiensis, bt for short), and the nucleic acid sequences of the Cry1Ab, cry2Ab, and Cry1Fa genes may be optimized or otherwise altered to increase the stability and availability of transcripts in the transformed cells.
In some embodiments of the invention, the soybean cells, seeds or plants comprising transgenic soybean event LP086-3 comprise in their genome the nucleic acid sequence of SEQ ID NO. 1, SEQ ID NO. 5 at positions 606-16789 and SEQ ID NO. 2, or SEQ ID NO. 5, in that order.
The Lepidoptera, the academic name Lepidotera, including moths and butterflies, is one of the most abundant insect species of agricultural and forestry pests, such as cotton bollworms, spodoptera frugiperda, prodenia litura, spodoptera exigua, soybean food heartworms and the like.
The 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene may be isolated from agrobacterium tumefaciens (Agrobacterium tumefaciens sp.) CP4 strain and the polynucleotide encoding the EPSPS gene may be modified by optimizing codons or otherwise achieving the goal of increasing the stability and availability of transcripts in transformed cells. The 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) gene can also be used as a selectable marker gene.
The term "glyphosate" refers to N-phosphonomethylglycine and its salts, and treatment with a "glyphosate herbicide" refers to treatment with any herbicide formulation containing glyphosate. The rate of use of a glyphosate formulation is selected to achieve an effective biological dosage that does not exceed the skill of an ordinarily skilled artisan. Treatment of a field containing plant material derived from transgenic soybean event LP086-3 with any herbicide formulation containing glyphosate will control weed growth in the field and will not affect the growth or yield of plant material derived from transgenic soybean event LP 086-3.
The DNA construct is introduced into a plant using transformation methods including, but not limited to, agrobacterium-mediated transformation, gene gun transformation, and pollen tube channel transformation.
The agrobacterium-mediated transformation method is a common method for plant transformation. The foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, i.e., the T-DNA region. The vector is transformed into an agrobacterium cell, which is subsequently used to infect plant tissue, and the T-DNA region of the vector comprising exogenous DNA is inserted into the plant genome.
The gene gun transformation method is to bombard plant cells (particle-mediated biolistic transformation) with a vector containing exogenous DNA.
The pollen tube channel transformation method utilizes a natural pollen tube channel (also called pollen tube guiding tissue) formed after plant pollination to carry exogenous DNA into embryo sacs through a bead core channel.
After transformation, the transgenic plants must be regenerated from the transformed plant tissue and offspring with the exogenous DNA selected using appropriate markers.
A DNA construct is a combination of DNA molecules that are linked to one another to provide one or more expression cassettes. The DNA construct is in particular a plasmid capable of self-replication in bacterial cells and containing various restriction enzyme sites for the introduction of DNA molecules providing functional genetic elements, i.e. promoters, introns, leader sequences, coding sequences, 3' terminator regions and other sequences. The expression cassette contained in the DNA construct includes the genetic elements necessary to provide for transcription of messenger RNA, and can be designed for expression in prokaryotic or eukaryotic cells. The expression cassette of the invention is designed to be most specifically expressed in plant cells.
A transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one nucleic acid expression cassette containing a gene of interest, inserting into the plant genome by transgenic means to produce a plant population, regenerating the plant population, and selecting a particular plant having the characteristics of being inserted into a particular genomic locus. The term "event" refers to both the original transformant comprising the heterologous DNA and the progeny of the transformant. The term "event" also refers to the progeny of a sexual cross between a transformant and other species of individuals containing heterologous DNA, even after repeated backcrosses with a backcross parent, the inserted DNA and flanking genomic DNA from the transformant parent are present at the same chromosomal location in the hybrid progeny. The term "event" also refers to a DNA sequence from an original transformant that comprises an inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which DNA sequence is expected to be transferred into progeny resulting from sexual crossing of a parental line containing the inserted DNA (e.g., the original transformant and progeny resulting from its selfing) with a parental line not containing the inserted DNA, and which progeny received the inserted DNA comprising the gene of interest.
"recombinant" in the context of the present invention refers to forms of DNA and/or proteins and/or organisms that are not normally found in nature and are therefore produced by manual intervention. Such manual intervention may result in recombinant DNA molecules and/or recombinant plants. The "recombinant DNA molecule" is obtained by artificially combining two otherwise isolated sequence segments, for example by chemical synthesis or by manipulation of isolated nucleic acid segments by genetic engineering techniques. Techniques for performing nucleic acid manipulations are well known.
The term "transgene" includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is altered by the presence of a heterologous nucleic acid, and includes the transgene originally so altered as well as progeny individuals produced from the original transgene by sexual crosses or asexual propagation. In the present invention, the term "transgene" does not include genomic (chromosomal or extrachromosomal) alterations by conventional plant breeding methods or naturally occurring events such as random allofertilisation, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
By "heterologous" in the present invention is meant that the first molecule is not normally found in combination with the second molecule in nature. For example, a molecule may originate from a first species and be inserted into the genome of a second species. Such molecules are thus heterologous to the host and are artificially introduced into the genome of the host cell.
Culturing transgenic soybean event LP086-3 that is resistant to lepidopteran insects and tolerant to glyphosate herbicide can be accomplished by the steps of: first sexually crossing a first parent soybean plant consisting of a soybean plant grown from transgenic soybean event LP086-3 and its progeny obtained by transformation with an expression cassette of the invention that is resistant to lepidopteran insects and tolerant to glyphosate herbicide, with a second parent soybean plant lacking resistance to lepidopteran insects and/or tolerant to glyphosate herbicide, thereby producing a multiplicity of first generation progeny plants; the progeny plants that are resistant to attack by lepidopteran insects and/or tolerant to glyphosate herbicide are then selected, and soybean plants that are resistant to lepidopteran insects and tolerant to glyphosate herbicide can be grown. These steps may further comprise backcrossing a progeny plant that is lepidopteran insect resistant and/or glyphosate tolerant with the second parent soybean plant or the third parent soybean plant, and selecting the progeny by infestation with a lepidopteran insect, application of a glyphosate herbicide, or by identification of a molecular marker associated with the trait (e.g., a DNA molecule comprising the junction site identified at the 5 'and 3' ends of the insertion sequence in transgenic soybean event LP 086-3), thereby producing a soybean plant that is lepidopteran insect resistant and tolerant to a glyphosate herbicide.
It will also be appreciated that two different transgenic plants can also be crossed to produce offspring containing two independent, separately added exogenous genes. Selfing of appropriate offspring can result in offspring plants that are homozygous for both added exogenous genes. Backcrossing of parent plants and outcrossing with non-transgenic plants as previously described are also contemplated, as are asexual propagation.
The term "probe" is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule, e.g., a radioisotope, ligand, chemiluminescent agent, or enzyme, can be attached. Such a probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one strand of DNA from the genome of transgenic soybean event LP086-3, whether the genomic DNA is from transgenic soybean event LP086-3 or seed or plant or seed or extract derived from transgenic soybean event LP 086-3. Probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.
The term "primer" is an isolated nucleic acid molecule that binds to a complementary target DNA strand by nucleic acid hybridization, anneals to form a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pairs of the invention relate to their use in the amplification of a target nucleic acid sequence, for example, by the Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.
Methods of designing and using primers and probes are well known in the art. The DNA molecules comprising the full length or fragments of SEQ ID NOS: 1-7 can be used as primers and probes for detecting soybean event LP086-3 and can be readily designed by one skilled in the art using the sequences provided herein.
The length of the probes and primers is generally 11 polynucleotides or more, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, and most preferably 30 polynucleotides or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes other than the target DNA sequence and maintaining hybridization ability to the target DNA sequence can be designed by conventional methods, it is preferred that the probes and primers of the present invention have complete DNA sequence identity to a contiguous nucleic acid of the target sequence.
Primers and probes based on the flanking genomic DNA and insert sequences of the invention may be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic soybean event LP086-3 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert sequence and soybean genome flanking regions, and fragments of the DNA molecule may be used as primers or probes.
The nucleic acid probes and primers of the invention hybridize to a target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic soybean event LP086-3 in a sample. The nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain conditions. As used herein, two nucleic acid molecules can be said to specifically hybridize to each other if they are capable of forming antiparallel double-stranded nucleic acid structures. Two nucleic acid molecules are said to be "complements" of one nucleic acid molecule if they exhibit complete complementarity. As used herein, a nucleic acid molecule is said to exhibit "complete complementarity" when each nucleotide of the two molecules is complementary to a corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "highly stringent" conditions. Deviations from complete complementarity are permissible provided that such deviations do not completely prevent the formation of double-stranded structures by the two molecules. In order to enable a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
As used herein, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing under highly stringent conditions to the complementary strand of a matching other nucleic acid molecule. Suitable stringent conditions for promoting DNA hybridization, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45℃followed by washing with 2.0 XSSC at 50℃are well known to those skilled in the art. For example, the salt concentration in the washing step may be selected from about 2.0 XSSC at low stringency conditions, about 0.2 XSSC at 50℃to high stringency conditions, about 50 ℃. In addition, the temperature conditions in the washing step may be raised from about 22 ℃ at room temperature under low stringency conditions to about 65 ℃ under high stringency conditions. The temperature conditions and salt concentration may both be varied, or one may remain unchanged while the other variable is varied. In particular, a nucleic acid molecule of the invention may specifically hybridize under moderately stringent conditions, e.g., at about 2.0 XSSC and about 65℃to one or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, or to a complement thereof, or to any fragment of the foregoing. More specifically, a nucleic acid molecule of the invention hybridizes specifically under highly stringent conditions to one or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, or to the complement thereof, or to any fragment of the above sequences. In the present invention, preferred marker nucleic acid molecules have SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 7 or a sequence complementary thereto, or a fragment of any of the above sequences. Another preferred marker nucleic acid molecule of the invention has 80% to 100% or 90% to 100% sequence identity with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 7 or the complement thereof, or any fragment of the above sequences. SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 7 can be used as markers in plant breeding methods to identify offspring of genetic crosses. Hybridization of the probe to the target DNA molecule may be detected by any method known to those skilled in the art, including, but not limited to, fluorescent labels, radiolabels, antibody-based labels, and chemiluminescent labels.
With respect to amplification (e.g., by PCR) of a target nucleic acid sequence using specific amplification primers, "stringent conditions" refer to conditions that allow hybridization of only the primer pair to the target nucleic acid sequence in a DNA thermal amplification reaction, and primers having a wild-type sequence (or its complement) corresponding to the target nucleic acid sequence are capable of binding to the target nucleic acid sequence and preferably produce a unique amplification product, i.e., an amplicon.
The term "specific binding (target sequence)" means that under stringent hybridization conditions, the probe or primer hybridizes only to the target sequence in a sample containing the target sequence.
As used herein, "amplified DNA," "amplification product," or "amplicon" refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a soybean plant is produced by sexual hybridization from a soybean sample containing the transgenic soybean event LP086-3 of the invention, or whether a soybean sample collected from a field contains the transgenic soybean event LP086-3, or a soybean extract, such as meal, flour, or oil, contains the transgenic soybean event LP086-3, DNA extracted from a soybean plant tissue sample or extract can be amplified by a nucleic acid amplification method using a primer pair to produce an amplicon diagnostic for the presence of DNA of the transgenic soybean event LP 086-3. The primer pair includes a first primer derived from a flanking sequence in the genome of the plant adjacent to the insertion site of the inserted foreign DNA, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic soybean event LP 086-3. The length of the amplicon may range from the combined length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred fifty nucleotide base pairs, and most preferably plus about four hundred fifty nucleotide base pairs or more.
Alternatively, the primer pair may be derived from flanking genomic sequences flanking the inserted DNA to produce an amplicon comprising the entire inserted nucleic acid sequence. One of the primer pairs derived from the plant genomic sequence may be located at a distance from the inserted DNA sequence that may range from one nucleotide base pair to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in the DNA thermal amplification reaction.
The nucleic acid amplification reaction may be accomplished by any nucleic acid amplification reaction method known in the art, including the Polymerase Chain Reaction (PCR). Various methods of nucleic acid amplification are well known to those skilled in the art. PCR amplification methods have been developed to amplify 22kb genomic DNA and 42kb phage DNA. These methods, as well as other DNA amplification methods in the art, may be used in the present invention. The inserted exogenous DNA sequence and flanking DNA sequences from transgenic soybean event LP086-3 can be obtained by amplifying the genome of transgenic soybean event LP086-3 with the provided primer sequences, and standard DNA sequencing of the PCR amplicon or cloned DNA after amplification.
DNA detection kits based on DNA amplification methods may contain DNA primer molecules that specifically hybridize to target DNA and amplify diagnostic amplicons under appropriate reaction conditions. The kit may provide agarose gel-based detection methods or a number of methods known in the art for detecting diagnostic amplicons. Kits comprising DNA primers homologous or complementary to any portion of the soybean genomic region of SEQ ID NO. 3 or SEQ ID NO. 4 and homologous or complementary to any portion of the transgene insertion region of SEQ ID NO. 5 are provided by the invention. In particular, primer pairs identified as useful in DNA amplification methods are SEQ ID NO. 8 and SEQ ID NO. 9, which amplify a diagnostic amplicon homologous to a portion of the 5' transgene/genomic region of transgenic soybean event LP086-3, wherein the amplicon comprises SEQ ID NO. 1. Other DNA molecules used as DNA primers may be selected from SEQ ID NO. 5.
Amplicons produced by these methods can be detected by a variety of techniques. One of the methods is Genetic Bit Analysis, which designs a DNA oligonucleotide strand that spans the insert DNA sequence and adjacent flanking genomic DNA sequences. The oligonucleotide strand is immobilized in a microwell of a microwell plate, and after PCR amplification of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product can hybridize to the immobilized oligonucleotide strand and serve as a template for a single base extension reaction using DNA polymerase and ddNTPs specifically labeled for the next desired base. The results may be obtained by fluorescence or ELISA-like methods. The signal represents the presence of an insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.
Another method is Pyrosequencing technology. The method contemplates an oligonucleotide strand spanning the insertion DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand and the single stranded PCR product of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences) are hybridized and then incubated with DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine-5' -phosphosulfate and luciferin. dNTPs are added separately and the resulting optical signal is measured. The optical signal represents the presence of an insertion/flanking sequence, which indicates that amplification, hybridization, and single base or multiple base extension reactions were successful.
Fluorescence polarization as described by Chen et al (Genome Res.) 9:492-498, 1999) is also one method that may be used to detect the amplicons of the present invention. The use of this method requires the design of an oligonucleotide strand spanning the insertion DNA sequence and adjacent genomic DNA binding sites. The oligonucleotide strand is hybridized to a single stranded PCR product of the target region (using one primer in each of the insert sequence and adjacent flanking genomic sequences) and then incubated with DNA polymerase and a fluorescent-labeled ddNTPs. Single base extension results in insertion of ddNTPs. Such an insertion can measure the change in its polarization using a fluorometer. The change in polarization represents the presence of an insertion/flanking sequence, which indicates that amplification, hybridization, and single base extension reactions were successful.
Taqman is described as a method for detecting and quantifying the presence of a DNA sequence, which is described in detail in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed that spans the intervening DNA sequence and adjacent genomic flanking binding sites, as described below. The FRET probe and PCR primers (one primer in each of the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent moiety and the quencher moiety on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal is representative of the presence of the insertion/flanking sequences, which indicates that amplification and hybridization were successful.
Suitable techniques for detecting plant material derived from transgenic soybean event LP086-3 based on hybridization principles may also include Southern blot hybridization, northern blot hybridization, and in situ hybridization. In particular, the suitable technique includes incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized. The detection method is dependent on the type of label attached to the probe, for example, radiolabeled probes can be detected by X-ray exposure and development, or enzymatically labeled probes can be detected by substrate conversion to effect a color change.
Tyangi et al (Nat. Biotech.) 14:303-308, 1996) describe the use of molecular markers in sequence detection. Briefly described, a FRET oligonucleotide probe is designed that spans the intervening DNA sequence and adjacent genomic flanking binding sites. The unique structure of the FRET probe results in it containing a secondary structure that is capable of retaining both the fluorescent moiety and the quenching moiety in close proximity. The FRET probe and PCR primers (one primer in each of the insert sequence and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Upon successful PCR amplification, hybridization of the FRET probe to the target sequence results in a loss of secondary structure of the probe, thereby spatially separating the fluorescent moiety from the quenching moiety, producing a fluorescent signal. The generation of a fluorescent signal is representative of the presence of the insertion/flanking sequences, which indicates that amplification and hybridization were successful.
Other described methods, such as microfluidics (microfluidics), provide methods and apparatus for isolating and amplifying DNA samples. The photodyes are used to detect and determine specific DNA molecules. A nano tube (nano tube) device comprising an electronic sensor for detecting DNA molecules or a nano bead binding to a specific DNA molecule and thus being detectable is useful for detecting the DNA molecules of the invention.
DNA detection kits may be developed using the compositions of the present invention and methods described in or known to the DNA detection arts. The kit is useful for identifying the presence or absence of DNA from transgenic soybean event LP086-3 in a sample, and can also be used to cultivate soybean plants containing DNA from transgenic soybean event LP 086-3. The kit may contain DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO. 1, 2, 3, 4 or 5, or other DNA primers or probes homologous to or complementary to DNA contained in the transgenic genetic element of DNA, which DNA sequences may be used in DNA amplification reactions or as probes in DNA hybridization methods.
The DNA structure of the transgene insert sequence contained in the soybean genome and illustrated in fig. 1 and table 1 at the soybean genome binding site comprises: a soybean LP086-3 flanking genomic region at the 5' end of the transgene insert, a portion of the insert from the right border Region (RB) of agrobacterium, the first expression cassette consisting of the arabidopsis ubiquitin gene promoter Ubi (pAtUbi 10), operably linked to the insect resistance gene Cry1Fa (Cry 1 Fa) of bacillus thuringiensis, operably linked to the terminator thof 25poly a from agrobacterium tumefaciens pTi 15955; the second expression cassette consisted of an arabidopsis actin 2 promoter (pAtAct 2), operably linked to an arabidopsis chloroplast transit peptide (spattp 2), operably linked to a bacillus thuringiensis insect-resistant Cry2Ab protein (Cry 2 Ab), and operably linked to a nopaline synthase transcription terminator (tNos); the third expression cassette is composed of the promoter and leader sequence of the Arabidopsis thaliana coding small subunit of 1, 5-bisphosphate carboxylase/oxygenase (pAtRBCS 4) E9 Arabidopsis gene promoter operably linked to the signal peptide sequence of the Arabidopsis thaliana coding small subunit of 1, 5-bisphosphate carboxylase/oxygenase (spatRbcS 4) operably linked to the insect-resistant Cry1Ab protein of Bacillus thuringiensis (Cry 1 Ab), operably linked to the transcription terminator of the 3' UTR sequence of the PT1 gene derived from the alfalfa encoding phosphate transporter (tMtPt); a fourth expression cassette was operably linked to a promoter derived from the Tsf1 gene of the soybean-derived elongation factor EF-lalpha (pGm gTsf 1), to the Arabidopsis chloroplast transit peptide (spatCTP 2), to the glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the Agrobacterium CP4 strain, and to a 3 '-terminal non-transcribed sequence terminator (tPsE 9) of the E9 gene derived from the pea nucleosome 1, 5-bisphosphate carboxylase, to a portion of the insert sequence from the left border region (LB) of Agrobacterium, and to the soybean plant LP086-3 flanking genomic region (SEQ ID NO: 5) at the 3' -terminal end of the transgenic insert sequence. In the DNA amplification method, the DNA molecule as a primer may be any part derived from the transgene insert sequence in the transgenic soybean event LP086-3, or any part derived from the DNA region of the flanking soybean genome in the transgenic soybean event LP 086-3.
Transgenic soybean event LP086-3 can be combined with other transgenic soybean varieties, such as soybeans that are tolerant to herbicides (e.g., glufosinate, dicamba, etc.), or transgenic soybean varieties that carry other insect-resistant genes (e.g., scara, grub, diabrotica, etc.). Various combinations of all of these different transgenic events, when bred with transgenic soybean event LP086-3 of the present invention, can provide improved hybrid transgenic soybean varieties that are resistant to multiple pests and tolerant to multiple herbicides. These varieties may exhibit superior characteristics such as yield enhancement compared to non-transgenic varieties and transgenic varieties of single trait.
The present invention provides transgenic soybean event LP086-3, a nucleic acid sequence for detecting soybean plants comprising the event, and methods of detecting the same, transgenic soybean event LP086-3 being resistant to feeding damage by lepidopteran pests and tolerant to the phytotoxic effects of glyphosate-containing agricultural herbicides. The dual trait soybean plants express the Cry1Ab, cry2Ab, and Cry1Fa proteins of bacillus thuringiensis, which provide resistance to ingestion damage by lepidopteran pests (e.g., asparagus caterpillar, prodenia litura, soybean borer, spodoptera frugiperda); and which expresses a glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein of agrobacterium strain CP4, which confers on plants tolerance to glyphosate.
Drawings
FIG. 1 is a schematic diagram showing the structure of a transgene insert sequence and a soybean genome binding site of the nucleic acid sequence for detecting soybean plant LP086-3 and the detection method thereof according to the present invention;
FIG. 2 is a schematic diagram showing the structure of a recombinant expression vector pLP086 for detecting the nucleic acid sequence of soybean plant LP086-3 and the detection method thereof;
FIG. 3 is an in vitro resistance effect of transgenic soybeans comprising transgenic soybean event LP086-3 of the present invention against lepidopteran pests;
FIG. 4 is a graph of the artificial insemination effect of transgenic soybeans comprising transgenic soybean event LP086-3 of the present invention in a cotton bollworm field;
FIG. 5 is a graph showing the effect of transgenic soybean comprising transgenic soybean event LP086-3 of the present invention on spodoptera exigua naturally occurring conditions;
FIG. 6 is a graph of the field effect of transgenic soybeans comprising transgenic soybean event LP086-3 of the present invention under spodoptera frugiperda naturally occurring conditions;
FIG. 7 is a plot of the field effect of the recommended spray concentration of transgenic soybeans comprising transgenic soybean event LP086-3 of the present invention in a field sprayed with 4-fold doses of glyphosate herbicide.
Detailed Description
The following is a further explanation of the nucleic acid sequence and the detection method of the present invention for detecting soybean plants LP086-3 by specific examples.
EXAMPLE 1 cloning and transformation
1.1 vector cloning
Recombinant expression vector pLP086 (shown in figure 2) was constructed using standard gene cloning techniques. The vector pLP086 comprises 4 transgene expression cassettes in tandem, the first expression cassette consisting of the arabidopsis ubiquitin gene promoter Ubi (pAtUbi 10), operably linked to the insect resistance gene Cry1Fa (Cry 1 Fa) of bacillus thuringiensis, operably linked to the terminator tx orf25poly a from agrobacterium tumefaciens pTi 15955; the second expression cassette consisted of an arabidopsis actin 2 promoter (pAtAct 2), operably linked to an arabidopsis chloroplast transit peptide (spattp 2), operably linked to a bacillus thuringiensis insect-resistant Cry2Ab protein (Cry 2 Ab), and operably linked to a nopaline synthase transcription terminator (tNos); the third expression cassette is composed of the promoter and leader sequence of the Arabidopsis thaliana coding small subunit of 1, 5-bisphosphate carboxylase/oxygenase (pATRbcS 4) E9 Arabidopsis gene promoter operably linked to the signal peptide sequence of the Arabidopsis thaliana coding small subunit of 1, 5-bisphosphate carboxylase/oxygenase (spatRbcS 4) operably linked to the insect-resistant Cry1Ab protein of Bacillus thuringiensis (Cry 1 Ab), operably linked to the 3' UTR sequence transcription terminator (tMtPt) of the PT1 gene from the alfalfa coding phosphate transporter; a fourth expression cassette was operably linked to the Arabidopsis chloroplast transit peptide (spatP 2), to the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the Agrobacterium CP4 strain, and to the 3 '-non-transcribed sequence terminator (tPsE 9) of the 3' -non-transcribed sequence terminator of the E9 gene derived from the pea nucleosome 1, 5-bisphosphate carboxylase small subunit (RbcS 2). The vector pLP086 was transformed into Agrobacterium LBA4404 (Invitrogen, chicago, USA; cat. No. 18313-015) by liquid nitrogen method, and the transformed cells were screened using 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) as a selection marker.
1.2 plant transformation
Transformation was performed using conventional agrobacterium infection, and aseptically cultured soybean (soybean variety JACK) young embryos were co-cultured with agrobacterium as described in this example 1.1 to transfer T-DNA in the constructed recombinant expression vector pLP086 into the soybean genome to generate transgenic soybean events.
For agrobacterium-mediated soybean transformation, briefly, immature chick embryos are isolated from soybeans, and the chick embryos are contacted with an agrobacterium suspension, wherein the agrobacterium is capable of transferring the nucleic acid sequences of cry1Ab, cry2Ab, cry1Fa genes and the nucleic acid sequences of epsps genes to at least one cell of one of the chick embryos (step 1: an infestation step) in which the young embryos are immersed in a suspension of agrobacteria (OD 660 = 0.4-0.6), in a infestation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, ph 5.3) to initiate inoculation, in a period of time (3 days) after the infestation step (step 2: co-cultivation step) in particular, the young embryos are cultivated after the infestation step on a solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, agar 8g/L, ph 5.8) in a period of co-cultivation in which the young embryos can be recovered in a selective recovery medium (MS salt 4.3 g/3 mg, 2, 35 g/L) of 2, 4-dichlorophenoxyacetic acid, pH 5.8) at least one antibiotic known to inhibit the growth of agrobacterium (cephalosporin), without the addition of a selection agent for plant transformants (step 3: and (5) a recovery step). Specifically, young embryos are cultured on solid medium with antibiotics but no selection agent to eliminate agrobacterium and provide a recovery period for the infected cells. The inoculated chick embryos are then cultured on a medium containing a selection agent (N- (phosphonomethyl) glycine) and the growing transformed calli are selected (step 4: selection step). Specifically, the young embryos are cultured on a selective solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, N- (phosphonomethyl) glycine 0.25mol/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, plant gel 3g/L, pH 5.8) with a selective agent, resulting in selective growth of the transformed cells. Then, the callus is regenerated into plants (step 5: regeneration step), specifically, the callus grown on the medium containing the selection agent is cultured on solid medium (MS differentiation medium and MS rooting medium) to regenerate the plants.
The selected resistant calli were transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, N- (phosphonomethyl) glycine 0.125mol/L, plant gel 3g/L, pH=5.8) and cultured at 25 ℃. The differentiated plantlets were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, agar 8g/L, pH=5.8), cultured to about 10cm high at 25℃and transferred to a greenhouse for cultivation until set. In the greenhouse, the cells were cultured at 28℃for 16 hours and at 20℃for 8 hours each day.
1.3 identification and screening of transgenic events
A total of 1500 independent transgenic T0 individuals were generated. Molecular detection (including target gene copy number, insertion position and the like), target character (insect resistance and herbicide resistance) and agronomic character evaluation are carried out on all T0 plants, and LP086-3 is obtained after abnormal transformant plants are removed.
Example 2 detection of transgenic Soybean event LP086-3 with TaqMan
About 100mg of leaves of transgenic soybean event LP086-3 was taken as a sample, genomic DNA thereof was extracted by Qiagen's DNeasy Plant Maxi Kit, and the copy numbers of cry1Ab, cry2Ab, cry1Fa and epsps were detected by Taqman probe fluorescent quantitative PCR method. Meanwhile, wild type soybean plants (non-transgenic, transformed recipients) were used as a control, and the detection and analysis were performed as described above. Experiments were repeated 3 times and averaged.
The specific method comprises the following steps:
step 11, taking 100mg of leaves of transgenic soybean event LP086-3, grinding into homogenate in a mortar by using liquid nitrogen, and taking 3 repeats of each sample;
step 12, extracting genomic DNA of the sample by using DNeasy Plant Mini Kit of Qiagen, wherein the specific method refers to the product instruction;
step 13, determining the concentration of the genomic DNA of the sample by using NanoDrop 2000 (Thermo Scientific);
step 14, adjusting the concentration of the genomic DNA of the sample to the same concentration value, wherein the concentration value ranges from 80 ng/mu l to 100 ng/mu l;
step 15, identifying the copy number of the sample by adopting a Taqman probe fluorescent quantitative PCR method, taking the sample with the identified known copy number as a standard substance, taking the sample of a wild type soybean plant (non-transgene, transformation receptor) as a control, repeating 3 times for each sample, and taking the average value; the fluorescent quantitative PCR primer and the probe sequences are respectively as follows:
the following primers and probes were used to detect cry1Ab gene sequence:
primer 1: TGGGAGGACGGAATGATATTG, as shown in SEQ ID NO. 16 of the sequence Listing;
primer 2: AACTCGTCCGTGAGCATCATC, as shown in SEQ ID NO. 17 of the sequence Listing;
probe 1: AACTCCGCGCTGCGATGAATCC, as shown in SEQ ID NO. 18 of the sequence Listing;
The following primers and probes were used to detect cry2Ab gene sequence:
primer 3: GGACAGAGGCACCGCATT, as shown in SEQ ID NO 19 of the sequence Listing;
primer 4: CGGGTCTGCAAGCAAACG, as shown in SEQ ID NO. 20 of the sequence Listing;
probe 2: TCCACTTGGCGGTTGAACTCCTCC, as shown in SEQ ID NO. 21 of the sequence Listing;
the following primers and probes were used to detect cry1Fa gene sequence:
primer 5: GCTATGTCCAGTCCCCAACCT, as shown in SEQ ID NO. 22 of the sequence Listing;
primer 6: CAAGCTGCTAACCTGCACTTGT, as shown in SEQ ID NO. 23 of the sequence Listing;
probe 3: CCCAAACGACACAGCGTCGCG, as shown in SEQ ID NO. 24 of the sequence Listing;
the following primers and probes were used to detect the epsps gene sequence:
primer 7: GCAAATCCTCTGGCCTTTCC, as shown in SEQ ID NO. 25 of the sequence Listing;
primer 8: TGAAGGACCGGTGGGAGAT, as shown in SEQ ID NO. 26 of the sequence Listing;
probe 4: CGTCCGCATTCCCGGCGA, as shown in SEQ ID NO 27 of the sequence Listing;
the PCR reaction system is that
The 50 Xprimer/probe mixture contained 45. Mu.L of each primer at a concentration of 1mM, 50. Mu.L of probe at a concentration of 100. Mu.M and 860. Mu.L of 1 XTE buffer, and was stored in amber tubes at 4 ℃.
The PCR reaction conditions were
The data were analyzed using SDS2.3 software (Applied Biosystems) to obtain a single copy of transgenic soybean event LP086-3.
Example 3 transgenic soybean event LP086-3 detection
3.1 genomic DNA extraction
DNA extraction according to the conventionally employed CTAB (cetyltrimethylammonium bromide) method: 2 g of tender transgenic soybean event LP086-3 leaves are ground into powder in liquid nitrogen, 0.5mL of DNA preheated at 65 ℃ is added to extract CTAB Buffer [20g/L CTAB,1.4M NaCl,100mM Tris-HCl,20mM EDTA (ethylenediamine tetraacetic acid) ], naOH is used for regulating the pH to 8.0, and the mixture is fully and uniformly mixed and extracted for 90min at 65 ℃; adding 0.5 volume of phenol and 0.5 volume of chloroform, and mixing the mixture upside down; centrifuging at 12000rpm for 10min; sucking the supernatant, adding 1-time volume of isopropanol, gently shaking the centrifuge tube, and standing at-20deg.C for 30min; further centrifuging at 12000rpm for 10min; collecting DNA to the bottom of the tube; discarding the supernatant, washing the precipitate with 0.5mL of 70% ethanol by volume; centrifuging at 12000rpm for 5min; vacuum pumping or blow-drying in an ultra clean bench; the DNA precipitate was dissolved in an appropriate amount of TE buffer (10 mM Tris-HCl,1mM EDTA,pH 8.0), and stored at a temperature of-20 ℃.
3.2 analysis of flanking DNA sequences
And (3) carrying out concentration measurement on the extracted DNA sample, so that the concentration of the sample to be measured is between 80 and 100 ng/. Mu.L. Genomic DNA was digested with selected restriction enzymes SpeI, pstI, bssHII (5 'end assay) and SacI, kpnI, xmaI, nheI (3' end assay), respectively. 26.5. Mu.L of genomic DNA, 0.5. Mu.L of the above-selected restriction enzyme and 3. Mu.L of the cleavage buffer were added to each cleavage system, and the cleavage was performed at an appropriate temperature for 1 hour. After the completion of the digestion, 70. Mu.L of absolute ethyl alcohol was added to the digestion system, the mixture was ice-washed for 30min, centrifuged at 12000rpm for 7min, the supernatant was discarded, and the mixture was dried, followed by 8.5. Mu.L of double distilled water (ddH 2O), 1. Mu.L of 10 XT 4 Buffer and 0.5. Mu. L T4 ligase at 4℃overnight. PCR amplification was performed with a series of nested primers to isolate 5 'and 3' transgenes/genomic DNA. Specifically, the isolated 5' transgene/genomic DNA primer combination includes SEQ ID NO. 13, SEQ ID NO. 34 as a first primer, SEQ ID NO. 35, SEQ ID NO. 36 as a second primer, and SEQ ID NO. 13 as a sequencing primer. The isolated 3' transgene/genomic DNA primer combination included SEQ ID NO. 15, SEQ ID NO. 37 as the first primer, SEQ ID NO. 38, SEQ ID NO. 39 as the second primer, SEQ ID NO. 15 as the sequencing primer, and the PCR reaction conditions are shown in Table 3.
The resulting amplicons were electrophoresed on a 2.0% agarose Gel to isolate the PCR reaction, followed by isolation of the fragment of interest from the agarose matrix using the QIAquick Gel extraction kit (catalogue # 28704, qiagen Inc., valencia, CA). The purified PCR product is then sequenced (e.g., ABI prism 377, PE Biosystems, foster City, CA) and analyzed (e.g., DNASTAR sequence analysis software, DNASTAR inc., madison, WI).
The 5 'and 3' flanking sequences and the junction sequences were confirmed using standard PCR methods. The 5' flanking sequences and the junction sequences can be confirmed using SEQ ID NO. 8 or SEQ ID NO. 12 in combination with SEQ ID NO. 9, SEQ ID NO. 13 or SEQ ID NO. 34. The 3' flanking sequences and the junction sequences can be confirmed using SEQ ID NO. 11 or SEQ ID NO. 14 in combination with SEQ ID NO. 10, SEQ ID NO. 15 or SEQ ID NO. 37. The PCR reaction system and the amplification conditions are shown in tables 3 and 4. Those skilled in the art will appreciate that other primer sequences may be used to confirm flanking and junction sequences.
DNA sequencing of the PCR products provides DNA that can be used to design other DNA molecules as primers and probes for identification of soybean plants or seeds derived from transgenic soybean event LP 086-3.
It was found that the soybean genomic sequence is shown flanking the right border of the transgenic soybean event LP086-3 insert (5 'flanking sequence) at nucleotides 1-560 of SEQ ID NO. 5 and that the soybean genomic sequence is shown flanking the left border of the transgenic soybean event LP086-3 insert (3' flanking sequence) at nucleotides 16849-17158 of SEQ ID NO. 5. The 5 'junction sequence is set forth in SEQ ID NO. 1 and the 3' junction sequence is set forth in SEQ ID NO. 2.
3.3 PCR zygosity assay
The junction sequence is a relatively short polynucleotide molecule, which is a novel DNA sequence that is diagnostic for the DNA of transgenic soybean event LP086-3 when detected in a polynucleic acid detection assay. The binding sequence of SEQ ID NO. 1 includes the T-DNA RB region insertion site of transgenic soybean event LP086-3 and 11bp each on one side of the soybean genomic DNA insertion site, and the binding sequence of SEQ ID NO. 2 includes the T-DNA LB region insertion site of transgenic soybean event LP086-3 and 11bp each on the other side of the soybean genomic DNA insertion site. Longer or shorter polynucleotide binding sequences may be selected from SEQ ID NO. 3 or SEQ ID NO. 4. The junction sequences (5 'junction region SEQ ID NO:1, and 3' junction region SEQ ID NO: 2) are useful as DNA probes or as DNA primer molecules in DNA detection methods. The junction sequences SEQ ID NO. 6 and SEQ ID NO. 7 are also novel DNA sequences in transgenic soybean event LP086-3, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic soybean event LP086-3 DNA. The sequence of SEQ ID NO. 6 (nucleotides 595-1154 of SEQ ID NO. 3) spans the LP086 construct DNA sequence and the pAtUbi10 transcription promoter sequence, and the sequence of SEQ ID NO. 7 (nucleotides 1-521 of SEQ ID NO. 4) spans the tPsE9 transcription termination sequence and the LP086 construct DNA sequence.
Furthermore, the amplicon is generated by using primers from at least one of SEQ ID NO. 3 or SEQ ID NO. 4, which primers when used in the PCR method generate a diagnostic amplicon for transgenic soybean event LP 086-3.
Specifically, a PCR product is generated from the 5 'end of the transgenic insert that is a portion of genomic DNA flanking the 5' end of the T-DNA insert in the genome comprising plant material derived from transgenic soybean event LP 086-3. This PCR product contains SEQ ID NO 3. For PCR amplification, primers 11 (SEQ ID NO: 8) hybridizing to the genomic DNA sequence flanking the 5' -end of the transgene insert and primers 12 (SEQ ID NO: 9) paired therewith located in the transcription promoter sequence of the transgene pAtUbi10 were designed.
A PCR product is generated from the 3 'end of the transgenic insert comprising a portion of genomic DNA flanking the 3' end of the T-DNA insert in the genome of the plant material derived from transgenic soybean event LP 086-3. This PCR product contains SEQ ID NO. 4. For PCR amplification, primers 14 (SEQ ID NO: 11) hybridizing to the genomic DNA sequences flanking the 3 '-end of the transgene insert and primers 13 (SEQ ID NO: 10) of the tNos transcription termination sequence at the 3' -end of the insert were designed to pair with.
The DNA amplification conditions illustrated in tables 3 and 4 can be used in the PCR zygosity assay described above to generate the diagnostic amplicon of transgenic soybean event LP 086-3. Detection of the amplicon may be performed by using a Stratagene Robocycle, MJ Engine, perkin-Elmer9700 or Eppendorf Mastercycler Gradien thermocycler, or the like, or by methods and apparatus known to those skilled in the art.
TABLE 3 PCR step and reaction mixture conditions for identification of 5' transgenic insert/genome combination region for transgenic soybean event LP086-3
Table 4, perkin-Elmer9700 thermal cycler conditions
Mix gently, if there is no thermal cap on the thermocycler, 1-2 drops of mineral oil can be added above each reaction solution. PCR was performed on a thermal cycler of Stratagene Robocycler (Stratagene, la Jolla, calif.), MJ Engine (MJ R-Biorad, hercules, calif.), perkin-Elmer9700 (Perkin Elmer, boston, mass.) or Eppendorf Mastercycler Gradient (Eppendorf, hamburg, germany) using the above cycling parameters (Table 4). The MJ Engine or Eppendorf Mastercycler Gradient thermocycler should operate in a calculated mode. The Perkin-Elmer9700 thermocycler is operated with a ramp rate (ramp speed) set to a maximum value.
The experimental results show that: primers 11 and 12 (SEQ ID NOS: 8 and 9), which when used in the PCR reaction of transgenic soybean event LP086-3 genomic DNA, generate an amplified product of 1154bp fragment, and when used in the PCR reaction of non-transformed soybean genomic DNA and non-LP 086-3 genomic DNA, NO fragment is amplified; primers 13 and 14 (SEQ ID NOS: 10 and 11), when used in the PCR reaction of transgenic soybean event LP086-3 genomic DNA, produced an amplified product of 880bp fragment, when used in the PCR reaction of non-transformed soybean genomic DNA and non-LP 086-3 soybean genomic DNA, NO fragment was amplified.
The PCR zygosity assay can also be used to identify whether the material derived from transgenic soybean event LP086-3 is homozygous or heterozygous. Primer 15 (SEQ ID NO: 12), primer 16 (SEQ ID NO: 13) and primer 17 (SEQ ID NO: 14), or primer 16 (SEQ ID NO: 13), primer 17 (SEQ ID NO: 14) and primer 18 (SEQ ID NO: 15) are used in an amplification reaction to generate a diagnostic amplicon of transgenic soybean event LP 086-3. The DNA amplification conditions illustrated in tables 5 and 6 can be used in the above zygosity assay to generate the diagnostic amplicon of transgenic soybean event LP 086-3.
TABLE 5 reaction solution for measuring the bondability
TABLE 6 determination of the bondability Perkin-Elmer9700 thermal cycler conditions
PCR was performed on a thermal cycler of Stratagene Robocycler (Stratagene, la Jolla, calif.), MJ Engine (MJ R-Biorad, hercules, calif.), perkin-Elmer9700 (Perkin Elmer, boston, mass.) or Eppendorf Mastercycler Gradient (Eppendorf, hamburg, germany) using the above cycling parameters (Table 6). The MJ Engine or Eppendorf Mastercycler Gradient thermocycler should operate in a calculated mode. The Perkin-Elmer9700 thermocycler is operated with a ramp rate (ramp speed) set to a maximum value.
In the amplification reaction, the biological sample containing the template DNA contains DNA diagnostic for the presence of transgenic soybean event LP086-3 in the sample. Or the reaction will produce two different DNA amplicons from a biological sample containing DNA derived from the soybean genome that is heterozygous for the corresponding allele of the insert DNA present in transgenic soybean event LP 086-3. These two different amplicons would correspond to a first amplicon derived from the wild-type soybean genomic locus and a second amplicon diagnostic for the presence of transgenic soybean event LP086-3 DNA. Only a soybean DNA sample corresponding to a single amplicon of the second amplicon described for the heterozygous genome is generated, the presence of transgenic soybean event LP086-3 can be diagnostically determined in the sample, and the sample is generated from a soybean seed that is homozygous for the allele corresponding to the inserted DNA present in transgenic soybean plant LP 086-3.
It should be noted that the primer pair for transgenic soybean event LP086-3 was used to generate an amplicon diagnostic for transgenic soybean event LP086-3 genomic DNA. These primer pairs include, but are not limited to, primers 11 and 12 (SEQ ID NOS: 8 and 9), and primers 13 and 14 (SEQ ID NOS: 10 and 11) for use in the DNA amplification method. In addition, a control primer 9 and 10 (SEQ ID NO:28 and SEQ ID NO: 29) for amplifying the soybean endogenous gene was included as an intrinsic criterion of the reaction conditions. Analysis of the transgenic soybean event LP086-3 DNA extract samples should include a positive tissue DNA extract control for transgenic soybean event LP086-3, a negative DNA extract control derived from non-transgenic soybean event LP086-3 and a negative control that does not contain a template soybean DNA extract. In addition to these primer pairs, any primer pair from SEQ ID NO. 3 or SEQ ID NO. 4, or the complement thereof, which when used in a DNA amplification reaction, produces an amplicon comprising SEQ ID NO. 1 or SEQ ID NO. 2 that is diagnostic for tissue derived from transgenic event soybean plant LP086-3, respectively, may be used. The DNA amplification conditions illustrated in tables 4-6 can be used to generate diagnostic amplicons of transgenic soybean event LP086-3 using appropriate primer pairs. Extracts that are presumed to contain soybean plant or seed DNA comprising transgenic soybean event LP086-3, or products derived from transgenic soybean event LP086-3, that when tested in a DNA amplification method produce an amplicon diagnostic for transgenic soybean event LP086-3, can be used as templates for amplification to determine the presence or absence of transgenic soybean event LP086-3.
Example 4 detection of transgenic Soybean event LP086-3 by Southern blot hybridization
4.1 DNA extraction for Southern blot hybridization
Southern blot analysis was performed using T4, T5 generation homozygous transformation events. Approximately 5 to 10g of plant tissue was ground in liquid nitrogen using a mortar and pestle. Plant tissue was resuspended in 12.5mL extraction buffer a (0.2M Tris ph=8.0, 50mM EDTA,0.25M NaCl,0.1%v/v β -mercaptoethanol, 2.5% w/v polyvinylpyrrolidone) and centrifuged at 4000rpm for 10 min (2755 g). After discarding the supernatant, the pellet was resuspended in 2.5mL of extraction buffer B (0.2M Tris ph=8.0, 50mM EDTA,0.5M NaCl,1%v/v β -mercaptoethanol, 2.5% w/v polyvinylpyrrolidone, 3% myo-aminoacyl, 20% ethanol) and incubated for 30 min at 37 ℃. During the incubation period, the samples were mixed once with a sterile loop. After incubation, an equal volume of chloroform/isoamyl alcohol (24:1) was added, gently mixed by inversion and centrifuged at 4000rpm for 20 minutes. The aqueous layer was collected and centrifuged at 4000rpm for 5 minutes after the addition of 0.54 volume of isopropanol to precipitate the DNA. The supernatant was discarded and the DNA pellet was resuspended in 500. Mu.L TE. To degrade any RNA present, the DNA was incubated with 1. Mu.L of 30mg/mL RNAaseA for 30 min at 37℃and centrifuged at 4000rpm for 5 min, and the DNA was precipitated by centrifugation at 14000rpm for 10 min in the presence of 0.5 volumes of 7.5M ammonium acetate and 0.54 volumes of isopropanol. After discarding the supernatant, the pellet was washed with 500. Mu.L of 70% ethanol and dried and resuspended in 100. Mu.L TE.
4.2 restriction enzyme digestion
DNA concentrations were quantitatively detected using a spectrophotometer or fluorometer (using 1 xTAE and GelRED dyes). In a 100. Mu.L reaction system, 5. Mu.g of DNA was digested each time. Respectively digesting the genome DNA by using restriction endonucleases EcoRV and MfeI, and taking partial sequences of Cry2Ab and Cry1Ab on the T-DNA as probes; respectively digesting the genome DNA by using restriction enzymes AvrII and MfeI, and taking a partial sequence of Cry1Fa on the T-DNA as a probe; genomic DNA was digested with restriction enzymes EcoRV and AvrII, respectively, and a partial sequence of EPSPS on T-DNA was used as a probe. For each enzyme, the digestate was incubated at the appropriate temperature overnight. The samples were spun down to a volume of 30 μl using a vacuum centrifugal evaporative concentrator (speed vacuum).
4.3 gel electrophoresis
Bromophenol blue loading dye was added to each sample from this example 4.2, and each sample was loaded onto a 0.7% agarose gel containing ethidium bromide, electrophoretically separated in TBE electrophoresis buffer, and the gel was electrophoresed overnight at 20 volts.
The gel was washed in 0.25M HCl for 15 minutes to depurinate the DNA, then washed with water. Southern blot hybridization was set as follows: in the tray 20 thick dry blotting papers were placed, and 4 thin dry blotting papers were placed thereon. In 0.4M NaOH, 1 sheet of Bao Yinji paper was pre-moistened and placed on the paper stack, followed by 1 sheet of Hybond-N+ transfer film pre-moistened in 0.4M NaOH (Amersham Pharmacia Biotech, # RPN 303B). The gel is placed on top, ensuring that there are no bubbles between the gel and the membrane. 3 additional pre-soaked blotters were placed on top of the gel and the buffer tray was filled with 0.4M NaOH. The gel stack and the buffer disc were connected with a wick pre-immersed in 0.4M NaOH, and the DNA was transferred to the membrane. DNA transfer was performed at room temperature for about 4 hours. After transfer, the Hybond membranes were rinsed in 2 XSSC for 10 seconds and the DNA was bound to the membrane by UV cross-linking.
4.4 hybridization
PCR was used to amplify the appropriate DNA sequences for probe preparation. The DNA probes are SEQ ID NO. 30,SEQ ID NO:31,SEQ ID NO:32 and SEQ ID NO. 33, or are homologous or complementary to the sequence parts. 25ng of probe DNA was boiled in 45. Mu.L TE for 5 minutes, placed on ice for 7 minutes, and then transferred to a Rediprime II (Amersham Pharmacia Biotech, #RPN1633) tube. After adding 5. Mu.l of 32P-labeled dCTP to the Rediprime tube, the probe was incubated at 37℃for 15 minutes. The probe was purified by centrifugation through a microcentrifuge G-50 column (Amersham Pharmacia Biotech, # 27-5330-01) according to the manufacturer's instructions to remove unincorporated dNTPs. Probe activity was measured using a scintillation counter. The Hybond membrane was prehybridized by wetting it with 20mL of prewarmed Church prehybridization solution (500mM Na3P04,1mM EDTA,7%SDS,1%BSA) for 30 min at 65 ℃. The labeled probe was boiled for 5 minutes and placed on ice for 10 minutes. To the pre-hybridization buffer, an appropriate amount of probe (1 million counts per 1mL of pre-hybridization buffer) was added and hybridization was performed overnight at 65 ℃. The next day, hybridization buffer was discarded, and after rinsing with 20mL Church rinse solution 1 (40mM Na3P04,1mM EDTA,5% SDS,0.5% BSA), the membrane was washed in 150mL Church rinse solution 1 at 65℃for 20 minutes. This procedure was repeated 2 times with Church rinse solution 2 (40mM Na3P04,1mM EDTA,1%SDS). The membrane is exposed to a phosphor screen or X-ray film to detect the location of probe binding.
Two control samples were included on each Southern: (1) DNA from negative (untransformed) isolates that are used to identify any endogenous soybean sequences that can hybridize to the element-specific probe; (2) DNA from positive segregants, into which HindIII digested pLP086 was introduced in an amount equivalent to one copy number based on probe length, to demonstrate the sensitivity of the experiment when detecting single gene copies within the soybean genome.
The hybridization data provides corroborated evidence that supports TaqMan PCR analysis, i.e., soybean plant LP086-3 contains a single copy of Cry2Ab, cry1Fa, cry1Ab and EPSPS genes. With this Cry2Ab probe, ecoRV and MfeI enzymatic hydrolysis produced single bands of about 15.5kb and 13.9kb in size, respectively; using the Cry1Fa probe, mfeI and AvrII enzymatic hydrolysis produced single bands of about 13.9kb and 24.6kb, respectively; enzymatic cleavage with Cry1Ab probe, mfeI and EcoRV produced single bands of about 13.9kb and 15.5kb in size, respectively; using this EPSPS probe, ecoRV and AvrII enzymatic hydrolysis produced single bands of about 15.5kb and 28.4kb, respectively. This indicates that Cry1Ab, cry2Ab, cry1Fa, and one copy of EPSPS each are present in soybean conversion event LP 086-3.
Example 5 insect resistance detection
5.1 bioassay of Soybean plant LP086-3
Transgenic soybean event LP086-3 and wild type soybean plants (non-transgenic, transformed recipient control (CK-)) 2 plants were bioassay of soybean borer (Leguminivora glycinivorella), spodoptera frugiperda (Spodoptera frugiperda), spodoptera litura (Spodoptera litura), cotton bollworm (Helicoverpa armigera) and spodoptera exigua (Spodoptera exigua), respectively, as follows:
fresh leaves (V3-V4 period) of 2 plants of transgenic soybean event LP086-3 and wild type soybean plants (non-transgenic, transformed acceptor control (CK-)) were taken respectively, washed clean with sterile water and blotted dry with absorbent paper, then soybean leaves were de-vein and cut into strips of about 1cm x 2cm in size, 1-3 (leaf number was determined according to insect diet) pieces of the cut strips were placed on filter paper at the bottom of a circular plastic petri dish, the filter paper was wetted with distilled water, 10 artificially fed initially hatched larvae were inoculated into each petri dish, the petri dish was capped, and the photoperiod (light/dark) was 16: statistics were carried out after 5 days of standing under 8 conditions. The statistical mortality (mortality= (number of dead insects/number of test insects) ×100%) was identified as an antagonistic level, and the results are shown in table 7 and fig. 3.
Table 7, in vitro anti-insect bioassay results for transgenic soybean event LP 086-3-mortality (%)
5.2 determination of field insect-resistant Effect of transgenic Soybean event LP086-3
(1) Bollworm (Bowls)
And (3) artificial inoculation is carried out on transgenic soybean event LP086-3 in soybean seedling stage, the total inoculation is carried out for 2 times, 20 heads of artificially fed initially hatched larvae are inoculated in each soybean filament, and after 3 days of inoculation, the second inoculation is carried out, and the inoculation quantity is the same as the first inoculation. After 14-21 days of insect inoculation, leaf pest rate, the number of surviving larvae of each leaf and leaf pest length are investigated plant by plant. Investigation was usually started 14 days after insect inoculation, and if the harmful level of the negative control material (CK-) reached the sense or high sense, then the investigation was considered to be effective, and if the investigation was not properly postponed, but the corresponding level was not reached yet 21 days after insect inoculation, then the insect inoculation was considered to be ineffective. According to the leaf damage rate and the number of surviving larvae, the average value of the damage level of the cotton bollworms in the soybean seedling stage of each cell to the leaf is counted according to the standard of the table 8, and the resistance level of the soybean seedling stage to the cotton bollworms is judged according to the standard of the table 9. The results of resistance to cotton bollworms at the seedling stage of transgenic soybean event LP086-3 are shown in FIG. 4 and Table 10. The results indicate that transgenic soybean event LP086-3 has a higher level of resistance to cotton bollworms, and that leaf damage rate, larval survival number, and leaf damage level of transgenic soybean event LP086-3 are significantly lower than the transformation recipient control (CK-).
TABLE 8 grading Standard for the extent to which soybean leaves are damaged by Helicoverpa armigera
TABLE 9 evaluation criteria for resistance of soybean leaves to Heliothis armigera
TABLE 10 results of resistance of transgenic soybean event LP086-3 seedling stage to Helicoverpa armigera
(2) Beet armyworm
The field natural pest-sensing test of asparagus caterpillar was performed in the 3 months 2021 on transgenic soybean seeds in the cliff region of three-city, south-hainan province. After 10-15 days of initial pest occurrence, and when most of control (CK-) plants are damaged by 4-6-year-old larvae, the pest rate of asparagus caterpillar to soybean plants is investigated plant by plant. The results of resistance of transgenic soybean event LP086-3 to asparagus caterpillar are shown in FIG. 5 and Table 11. The results show that the pest rate of the spodoptera exigua on the transgenic soybean event LP086-3 is significantly reduced compared with the control (CK-) under the naturally occurring condition of the spodoptera exigua, thereby indicating that the transgenic soybean event LP086-3 has higher resistance to the spodoptera exigua.
Table 11 results of resistance to spodoptera exigua under Natural pest-sensing conditions for transgenic soybean event LP086-3
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(3) Spodoptera frugiperda (L.) kurz
The field natural pest-sensing test of spodoptera frugiperda was performed at 2021 at 3 months on a transgenic soybean planting base in the cliff region of three-city, south-hainan province. After 10-15 days of initial pest occurrence, and when the transformation receptor control (CK-) is mostly damage of 4-6-year-old larvae, the pest rate of spodoptera frugiperda to soybean plants is investigated strain by strain. The results of resistance of transgenic soybean event LP086-3 to Spodoptera frugiperda are shown in FIG. 6 and Table 12. The results show that the spodoptera littoralis has a significantly reduced rate of damage to the transgenic soybean event LP086-3 as compared to the control (CK-) under naturally occurring conditions of spodoptera littoralis, thereby demonstrating that the transgenic soybean event LP086-3 has a higher resistance to spodoptera littoralis.
Table 12 results of resistance of transgenic soybean event LP086-3 to Spodoptera frugiperda under natural insect-sensing conditions
(4) Prodenia litura (L.) DC
The field natural pest-sensing test of prodenia litura was performed at 2021 at 3 months in the planting base of transgenic soybean in the state of cliff in three-city, south-hainan province. And after 10-15 days of initial pest occurrence, and when the transformation receptor control (CK-) is mostly damage of 4-6-year-old larvae, the pest rate of the prodenia litura to soybean plants is investigated strain by strain. The results of resistance of transgenic soybean event LP086-3 to Spodoptera litura are shown in Table 13. The result shows that under the condition that the prodenia litura naturally occurs, compared with a control (CK-) the damage rate of the prodenia litura to the transgenic soybean event LP086-3 is obviously reduced, thereby indicating that the transgenic soybean event LP086-3 has higher resistance to the prodenia litura.
TABLE 13 results of resistance of transgenic soybean event LP086-3 to Twill moth under natural insect-sensing conditions
(5) Soybean borer
The field natural pest-sensing test of soybean borer was performed at 2021 at 3 months on a transgenic soybean planting base in the state of cliff in the three-city of hainan province. And after 10-15 days of initial pest occurrence, and when the transformation receptor control (CK-) is mostly damage of 4-6-year-old larvae, the pest rate of soybean borers to soybean plants is investigated strain by strain. The results of resistance of transgenic soybean event LP086-3 to soybean heartworm are shown in Table 14. The results show that under the condition that the soybean borer naturally occurs, the damage rate of the soybean borer to the transgenic soybean event LP086-3 is obviously reduced compared with the control (CK-) and therefore, the transgenic soybean event LP086-3 has higher resistance to the soybean borer.
TABLE 14 results of resistance of transgenic soybean event LP086-3 to soybean borer under natural pest-sensing conditions
Example 6 herbicide tolerance detection of soybean transformation event
The test selects the pesticide (41% glyphosate isopropyl ammonium salt aqua) for spraying. A random block design was used, 3 replicates. The area of the cell is 20m2 (5 m multiplied by 4 m), the seedling is 340, the conventional cultivation and management are carried out, and a 1m wide isolation belt is arranged between the cells. Transgenic soybean event LP086-3 and the transformation receptor control (CK-) were treated with 2 treatments, respectively: 1) Spraying clear water; 2) The pesticide was sprayed at 3360 g a.e./ha dose at the V3 leaf stage and then again at V8 stage at the same dose. It should be noted that the conversion of glyphosate herbicide of different content and dosage forms to equivalent amounts of glyphosate acid is applicable to the following conclusion. The phytotoxicity symptoms were investigated at 1 and 2 weeks after dosing, respectively, and the yield of the cells was determined at harvest. The phytotoxicity symptom grading criteria are shown in Table 15. The herbicide damage rate is used as an evaluation index to evaluate an index of herbicide tolerance of a transformation event, specifically, the herbicide damage rate (%) Σ (peer damage number×number of grades)/(total number×highest grade) ×100; the herbicide damage rate refers to the glyphosate damage rate, and the glyphosate damage rate is determined according to the phytotoxicity investigation result of 2 weeks after the glyphosate treatment. The soybean yield per cell was measured as the total yield (weight) of soybean grains in the middle 3 rows of each cell, and the yield difference between the different treatments was measured as a yield percentage (% yield=glyphosate sprayed yield/clear water sprayed yield×100. The results of transgenic soybean event LP086-3 for herbicide tolerance and soybean yield are shown in fig. 7 and table 16.
Table 15, grading Standard of the extent of phytotoxicity of glyphosate herbicide to Soybean
Table 16, results of transgenic Soybean event LP086-3 for tolerance to glyphosate herbicide and Soybean yield results
The results show that in terms of herbicide (glyphosate) damage: 1) The transgenic soybean event LP086-3 had a rate of victimization of substantially 0 under glyphosate herbicide (3360 g a.e./ha) treatment, whereby the transgenic soybean event LP086-3 had good glyphosate herbicide tolerance.
In terms of yield: the yield of the transgenic soybean event LP086-3 is not obviously different under the 2 treatments of spraying water and spraying 3360g a.e./ha glyphosate, and after the glyphosate herbicide is sprayed, the yield of the transgenic soybean event LP086-3 is slightly improved compared with the water spraying treatment, thereby further indicating that the transgenic soybean event LP086-3 has good glyphosate herbicide tolerance.
In summary, regenerated transgenic soybean plants were examined for the presence of cry1Ab, cry2Ab, cry1Fa and epsps genes by taqman analysis (see example 2) and characterized for insect resistance and copy number of glyphosate herbicide tolerant lines. Event LP086-3 was selected to be excellent by screening, with single copy transgenes, good insect resistance, glyphosate herbicide tolerance and excellent agronomic performance, based on copy number of the gene of interest, good insect resistance, glyphosate herbicide tolerance and agronomic performance (see example 5 and example 6).

Claims (7)

1. A nucleic acid molecule for detecting transgenic soybean event LP086-3, wherein the sequence of the nucleic acid molecule is shown in one or more of SEQ ID No. 1-2, SEQ ID No. 3-4, SEQ ID No. 5 or the complement thereof, the nucleic acid molecule is derived from transgenic soybean event LP086-3, and soybean seeds of transgenic soybean event LP086-3 have been deposited with the chinese collection of typical cultures under accession No. cctccc No. P202327.
2. A DNA primer pair comprising a first primer and a second primer, wherein when said first primer and said second primer are used in an amplification reaction with DNA containing soybean event LP086-3, an amplicon is produced that detects soybean event LP086-3 in a sample,
the first primer is selected from SEQ ID NO. 8 or SEQ ID NO. 12, and the second primer is selected from SEQ ID NO. 9 or SEQ ID NO. 13; or the first primer is selected from SEQ ID NO. 10 or SEQ ID NO. 15, and the second primer is selected from SEQ ID NO. 11 or SEQ ID NO. 14; the soybean seeds containing the soybean event LP086-3 are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: P202327.
3. A method of detecting the presence of DNA from transgenic soybean event LP086-3 in a sample, comprising:
(1) Contacting a sample to be detected with the DNA primer pair of claim 2 in a nucleic acid amplification reaction;
(2) Performing a nucleic acid amplification reaction;
(3) Detecting the presence of an amplification product;
the amplification product comprises a nucleic acid sequence of sequence SEQ ID NO. 1 or 2 or a complementary sequence thereof, namely DNA representing that the detection sample comprises transgenic soybean event LP 086-3; the soybean seeds containing the soybean event LP086-3 are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: P202327.
4. A DNA detection kit comprising: the DNA primer pair of claim 2.
5. A method of protecting a soybean plant from insect infestation comprising providing transgenic soybean plant cells in a diet of a target insect; target insects that ingest the transgenic soybean plant cells are inhibited from further ingest the transgenic soybean plants; the insect is lepidopteran insect; the seeds of the transgenic soybeans are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: P202327.
6. A method of protecting soybean plants from injury caused by herbicides, characterized by planting transgenic soybean plants; applying an effective dose of a glyphosate herbicide; the seeds of the transgenic soybeans are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: P202327.
7. A method of controlling weeds in a field where soybean plants are grown, comprising applying an effective dose of a glyphosate herbicide to the field where transgenic soybean plants are grown; the seeds of the transgenic soybeans are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: P202327.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106086011A (en) * 2016-06-18 2016-11-09 北京大北农科技集团股份有限公司 For detecting nucleotide sequence and the detection method thereof of herbicide tolerant bean plant DBN9004
CN111247255A (en) * 2019-08-09 2020-06-05 北京大北农生物技术有限公司 Nucleic acid sequence for detecting soybean plant DBN8007 and detection method thereof
CN114787389A (en) * 2022-02-21 2022-07-22 北京大北农生物技术有限公司 Nucleic acid sequence for detecting soybean plant DBN8205 and detection method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106086011A (en) * 2016-06-18 2016-11-09 北京大北农科技集团股份有限公司 For detecting nucleotide sequence and the detection method thereof of herbicide tolerant bean plant DBN9004
CN111247255A (en) * 2019-08-09 2020-06-05 北京大北农生物技术有限公司 Nucleic acid sequence for detecting soybean plant DBN8007 and detection method thereof
WO2021026689A1 (en) * 2019-08-09 2021-02-18 北京大北农生物技术有限公司 Nucleic acid sequence used for detecting soybean plant dbn8007 and detection method therefor
CN114787389A (en) * 2022-02-21 2022-07-22 北京大北农生物技术有限公司 Nucleic acid sequence for detecting soybean plant DBN8205 and detection method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Spatial Lipidomics of EPSPS and PAT Transgenic and Non-Transgenic Soybean Seeds Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging;Zhentao Ren et al.;J Agric Food Chem .;第71卷(第26期);第10190-10202页 *
转基因大豆研究及应用进展;崔宁波 等;西北农业学报;第25卷(第8期);第1111-1124页 *
转基因技术在大豆性状改良上的应用;杜艳丽 等;大豆科学;第34卷(第2期);第320-328页 *

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