CN116640880B - Transgenic soybean event JK1001-1 and detection method thereof - Google Patents
Transgenic soybean event JK1001-1 and detection method thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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 JK1001-1, a representative sample of seed comprising said event having been deposited under accession number CCTCC NO. P202332. The transgenic soybean event JK1001-1 of the invention has the following advantages: contains human lactoferrin gene and can stably express human lactoferrin; tolerance to the commonly used commercial herbicide glyphosate; the soybean yield is not reduced; molecular markers can be used 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 JK 1001-1.
Description
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 a transgenic soybean event JK1001-1 containing a human lactoferrin gene and applied to glyphosate herbicide tolerance, and a nucleic acid sequence and a method for detecting the transgenic soybean JK 1001-1.
Background
Lactoferrin is a protein widely distributed in tissues with secretory functions and its secretions, found in milk earliest in 1939, and isolated from milk and human milk in 1960. In the human bodyThe content of colostrum is highest, up to 8mg/ml, 1.5-4mg/ml in milk, 2mg/ml in tears, and lower in other secretions (such as saliva, joint fluid, blood plasma), generally lower than 0.01mg/ml. It is believed to be associated with iron transport and storage, belonging to the Lactotransferrin (Lactotransferrin) family. Lactoferrin sequences are highly conserved among human, bovine, murine, porcine, etc., glycoproteins of about 690 amino acid residues and molecular weights of about 76-81kDa. In three dimensions, lactoferrin comprises two N-and C-leaflets (lobes), each capable of binding a single Fe in the deep gap between the two domains 3+ Ions.
In the digestive tract, lactoferrin is hydrolyzed into a small peptide with physiological functions, namely lactoferrin active polypeptide (Lfcin B), which is a polypeptide containing 25 amino acids, has strong alkalinity (pH is more than 8.15), and has the characteristics of heat resistance, difficult degradation in the digestive tract, immune activity regulation, broad-spectrum bacteriostasis and the like, and two reverse beta-sheet structures of hydrophilic and lipophilic are formed in aqueous solution. This is also the basis for the activity of oral lactoferrin. To date, research, development and application of lactoferrin have become important points of scientific research, and the existing research shows that lactoferrin has activities of resisting bacteria, resisting viruses, resisting inflammation, resisting cancer, regulating immunity, promoting bone formation, regulating intestinal flora and the like, and is widely applied in the directions of disease prevention, auxiliary treatment and the like.
At present, the preparation of the lactoferrin mainly depends on the extraction from milk, for example, a flying crane group applies an advanced chromatographic ultrafiltration technology, thereby realizing the extraction of the lactoferrin and building a first lactoferrin automatic production line in industry. However, the extraction of lactoferrin from milk has the risk of animal pathogen contamination, and in addition, the natural content of lactoferrin is insufficient, and only 1g of protein can be extracted from about 14kg of milk, which limits the mass production of lactoferrin from milk, and also makes the price of lactoferrin as high as 7000-8000 yuan/kg.
For the mass production of lactoferrin, there are currently successful cases of expressing lactoferrin in escherichia coli, pichia pastoris, mice, cows, potatoes and rice, whereas proteins expressed by prokaryotes are usually not biologically active due to the lack of post-translational modifications. The expression of these proteins in yeast or animal cell culture is not only costly but also requires high conditions.
The plant fruits or seeds have stronger protein synthesis function, and if the transgenic plants can express the lactoferrin, the product is close to natural, i.e. has higher bioactivity, high expression quantity, low cost and simple and easy implementation. However, few in the prior art use plants, particularly soybeans, to produce lactoferrin in large quantities.
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. An important agronomic trait in soybean production is herbicide tolerance, particularly glyphosate tolerance. Tolerance of soybeans to glyphosate herbicide can be obtained by transgenic methods to express glyphosate herbicide tolerance genes (epsps) in soybean plants.
In addition to the functional gene itself, the choice of regulatory elements and their sequential arrangement are critical 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 contain the human lactoferrin gene and have excellent glyphosate herbicide resistance without affecting soybean yield, and transgenic traits can be backcrossed into other genetic backgrounds by crossing using conventional breeding methods. 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, contains stable human lactoferrin gene expression and has glyphosate herbicide resistance, so that the varieties have broad-spectrum weed control capability and can be well suitable for 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 JK1001-1, a nucleic acid sequence for detecting the event JK1001-1 of a soybean plant and a detection method thereof, which can accurately and rapidly identify whether a biological sample contains a DNA molecule of a specific transgenic soybean event JK 1001-1.
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 JK1001-1, and a representative sample of seed comprising the event has been deposited at the chinese typical culture collection (CCTCC, address: eight of 299 universities in the armed sector, marchand, hubei, university collection, zip code 430072) under accession number cctccc No. P202332 on day 29 of 2023: soybean (glycine max xl). In some embodiments, the nucleic acid sequence is an amplicon diagnostic for the presence of soybean event JK 1001-1.
In some embodiments of the invention, the invention provides a nucleic acid sequence comprising at least 12 consecutive nucleotides of SEQ ID NO. 3 or a complement thereof and/or at least 8 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 of 22 nucleotides in length, which is positioned near the insertion junction at the 5 '-end of the insertion sequence in the transgenic soybean event JK1001-1, 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 presence of the transgenic soybean event JK1001-1 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 of 22 nucleotides in length near the insertion junction at the 3 'end of the insertion sequence in transgenic soybean event JK1001-1, 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 presence of the transgenic soybean event JK1001-1 can be identified by the inclusion of the SEQ ID NO. 2 or the complementary sequence thereof.
The nucleic acid sequences provided by the invention may be at least 12 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 12 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 JK1001-1 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 the complementary sequence thereof is a sequence with the length of 1170 nucleotides, which is positioned near the insertion junction at the 5' -end of the insertion sequence in the transgenic soybean event JK1001-1, the SEQ ID NO. 3 or the complementary sequence thereof consists of a soybean flanking genomic DNA sequence with 600 nucleotides (nucleotides 1-600 of SEQ ID NO. 3), a construction RB terminal sequence with 383 nucleotides (nucleotides 601-983 of SEQ ID NO. 3) of the JK1001-1 construct, and a partial sequence prGmA1aB1b with 187 nucleotides (nucleotides 984-1170 of SEQ ID NO. 3), and the presence of the transgenic soybean event JK1001-1 can be identified by the SEQ ID NO. 3 or the complementary sequence thereof.
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 JK1001-1 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 SEQ ID NO. 4 or the complementary sequence thereof is 1092 nucleotides in length near the insertion junction in the 3' -end of the insertion sequence of transgenic soybean event JK1001-1, and consists of 137 nucleotides of the tPse9 partial sequence of the JK1001 construct (nucleotides 1-137 of SEQ ID NO. 4), 215 nucleotides of the LB terminal sequence (nucleotides 138-352 of SEQ ID NO. 4) and 740 nucleotides of the soybean integration site flanking genomic DNA sequence (nucleotides 353-1092 of SEQ ID NO. 4), and the presence of the transgenic soybean event JK1001-1 can be identified by including the SEQ ID NO. 4 or the complementary sequence thereof.
The SEQ ID NO. 5 or its complement is a sequence of 9822 nucleotides in length characterizing transgenic soybean event JK1001-1, which specifically comprises the genome and genetic elements shown in Table 1. The presence of transgenic soybean event JK1001-1 can be identified by comprising said SEQ ID NO. 5 or its complement.
Table 1, genome and genetic element contained in SEQ ID NO. 5
| Genetic element | Length of | At position on SEQ ID NO. 5 |
| 5' genome | 600bp | 1-600 |
| RB region | 383bp | 601-983 |
| prGmA1aB1b | 2202bp | 984-3185 |
| cGmhlF | 2151bp | 3192-5342 |
| tNos | 253bp | 5349-5601 |
| prGm17gTsf1 | 999bp | 5613-6611 |
| spAtCTP2 | 228bp | 6613-6840 |
| cEPSPS | 1368bp | 6841-8208 |
| tPse9 | 643bp | 8225-8867 |
| LB zone | 215bp | 8868-9082 |
| 3' genome | 740bp | 9083-9822 |
The nucleic acid sequence or its complement can be used in a DNA amplification method to produce an amplification product, the presence of transgenic soybean event JK1001-1 or its progeny in a biological sample being diagnosed by detection of the amplification product; the nucleic acid sequence or its complement can be used in a nucleotide assay to detect the presence of transgenic soybean event JK1001-1 or its progeny 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 JK1001-1, produces an amplification product that detects soybean event JK1001-1 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 12 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 8 consecutive nucleotides of SEQ ID NO. 4 or its complement.
Further, the amplification product comprises consecutive nucleotides 1 to 11 or 12 to 22 in SEQ ID NO. 1 or its complement, or consecutive nucleotides 1 to 11 or 12 to 22 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 12 contiguous nucleotides of SEQ ID NO. 3 or its complement, or at least 8 contiguous nucleotides of SEQ ID NO. 4 or its complement; further, the probe comprises continuous nucleotides at positions 1-11 or 12-22 in SEQ ID NO. 1 or the complementary sequence thereof, or continuous nucleotides at positions 1-11 or 12-22 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 12 consecutive nucleotides of SEQ ID NO. 3 or its complement, or at least 8 consecutive nucleotides of SEQ ID NO. 4 or its complement;
in some embodiments, the marker nucleic acid molecule comprises consecutive nucleotides 1-11 or 12-22 of SEQ ID NO. 1 or its complement, or consecutive nucleotides 1-11 or 12-22 of SEQ ID NO. 2 or its complement.
Further, the present invention provides a method of detecting the presence of DNA comprising transgenic soybean event JK1001-1 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 group consisting of the sequences SEQ ID NOs 1-7 or the complement thereof, i.e., is indicative of the presence of DNA comprising transgenic soybean event JK1001-1 in the test sample.
The invention also provides a method of detecting the presence of DNA comprising transgenic soybean event JK1001-1 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, herbicide tolerance is determined to be genetically linked to the marker nucleic acid molecule by marker-assisted breeding analysis.
The invention also provides a DNA detection kit, comprising: a DNA primer pair that produces an amplicon diagnostic for transgenic soybean event JK1001-1, 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 12 contiguous nucleotides of the homologous sequence of SEQ ID NO. 3 or the complement thereof, or at least 8 contiguous 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 JK1001-1 or its progeny.
Further, the DNA molecule comprises the continuous nucleotides at positions 1-11 or 12-22 in SEQ ID NO. 1 or the complementary sequence thereof, or the continuous nucleotides at positions 1-11 or 12-22 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 a nucleic acid sequence encoding human lactoferrin cGmhlF, a nucleic acid sequence encoding glyphosate herbicide tolerance EPSPS protein, the nucleic acid sequence of the specific region comprising the sequence shown as 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
| Sequence number (SEQ) ID NO） | DESCRIPTION OF THE SEQUENCES |
| 1 | RB end crossing junction sequence (containing partial T-DNA RB end sequence and genome sequence, 22 bp) |
| 2 | LB terminal cross junction sequence (containing partial T-DNA LB terminal sequence and genome sequence, 22 bp) |
| 3 | The nucleotide sequence located near the insertion binding site at the 5' -end of the insertion sequence is the RB-end (containing Has genome about 600bp, T-DNA570bp |
| 4 | The nucleotide sequence located near the insertion binding site at the 3' -end of the insertion sequence is the LB-end (containing Has genome 740bp sequence and T-DNA352bp |
| 5 | T-DNA full-length sequence (genome sequence of 600bp and 740bp extended from both ends of LB and RB respectively) |
| 6 | Sequence located inside SEQ ID NO. 3, JK1001-1T-DNA sequence |
| 7 | Sequence located inside SEQ ID NO. 4, JK1001-1T-DNA sequence |
| 8 | First primer for amplifying SEQ ID NO. 3, primer 5 |
| 9 | Second primer for amplifying SEQ ID NO. 3, primer 6 |
| 10 | First primer for amplifying SEQ ID NO. 4, primer 7 |
| 11 | Second primer for amplifying SEQ ID NO. 4, primer 8 |
| 12 | Primer on 5' flanking genome, primer 9 |
| 13 | Primer 10 on T-DNA paired with sequence 12 |
| 14 | Primer on 3' flanking genome, primer 11 |
| 15 | Primer 12 on T-DNA paired with sequence 14 |
| 16 | Taqman assay cgmhlF primer 1 |
| 17 | Taqman assay cgmhlF primer 2 |
| 18 | Taqman assay cgmhlF probe 1 |
| 19 | Taqman detection EPSPS primer 3 |
| 20 | Taqman detection EPSPS primer 4 |
| 21 | Taqman detection EPSPS probe 2 |
| 22 | Primer 15 located on the T-DNA, which hybridizes to SEQ ID NO:13 are consistent in direction |
| 23 | Primer 16 located on the T-DNA, which hybridizes to SEQ ID NO:13 opposite direction |
| 24 | Primer 17 located on the T-DNA, which hybridizes to SEQ ID NO:13 opposite direction |
| 25 | Primer 18 located on the T-DNA, which hybridizes to SEQ ID NO:15 direction is consistent |
| 26 | Primer 19 located on the T-DNA, which hybridizes to SEQ ID NO:15 opposite directions |
| 27 | Primer 20 located on the T-DNA, which hybridizes to SEQ ID NO:15 opposite directions |
| 28 | Soybean elongation factor gene Gm-EF1 alpha primer 13 |
| 29 | Soybean elongation factor gene Gm-EF1 alpha primer 14 |
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 sequences from 612 to 9071 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 a glyphosate herbicide to a field in which at least one transgenic soybean plant comprising transgenic soybean event JK1001-1 is grown.
The present invention also provides a method of controlling weeds in a field in which soybean plants are grown, comprising applying an effective dose of a glyphosate herbicide to the field in which at least one transgenic soybean plant is grown, said transgenic soybean plant comprising in sequence the nucleic acid sequence of SEQ ID NO. 1, SEQ ID NO. 5, positions 612-9071 and SEQ ID NO. 2; or the genome of the transgenic soybean plant comprises a sequence shown as SEQ ID NO. 5.
In some embodiments, the invention provides a method of culturing a glyphosate herbicide tolerant soybean plant comprising:
planting at least one soybean seed comprising transgenic soybean event JK 1001-1;
growing the soybean seeds into soybean plants;
spraying the soybean plants with an effective dose of glyphosate herbicide, and harvesting plants having reduced plant damage as compared to other plants not having the transgenic soybean event JK 1001-1.
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 JK1001-1 and selecting a soybean plant that is tolerant to glyphosate. In some embodiments, the method comprises: sexual crossing a transgenic soybean event JK1001-1 first parent soybean plant 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.
The present invention also provides a composition that results from transgenic soybean event JK1001-1, which is soybean meal, soybean oil, soybean protein, soybean meal, okara, and the like. In some embodiments, the composition may be soy flour, soy protein isolate, 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 JK1001-1 material in the composition. In particular, the compositions include, but are not limited to, soy flour, soy protein isolate, soy oil, soy protein, soy products, meal, any other food product to be consumed by animals as a food source, or otherwise as an ingredient of soy oil or stearic acid for food industry use, and the like.
The probe or primer pair-based detection methods and/or kits of the present invention can be employed to detect a transgenic soybean event JK1001-1 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 shown as 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 JK 1001-1.
In summary, the transgenic soybean event JK1001-1 of the invention has herbicide resistance and has the following advantages: 1) A human lactoferrin gene with stable genetic expression; 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, event JK1001-1 of the invention has lactoferrin expression level up to 2.08mg/g; the tolerance to glyphosate herbicide is high, and the plant can be protected under the condition of 4 times of recommended dosage, so that the damage rate is as low as 0%; and the agronomic characters of the plants containing the event are excellent, and the yield percentage can reach as high as 102 percent. Meanwhile, the primer or probe sequence provided in the detection method can generate an amplification product identified as the transgenic soybean event JK1001-1 or a progeny thereof, and can rapidly, accurately and stably identify the existence of plant materials derived from the transgenic soybean event JK 1001-1.
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 a transgenic soybean event designated JK1001-1 and its progeny, said transgenic soybean event JK1001-1 being soybean plant JK1001-1 comprising plants and seeds of transgenic soybean event JK1001-1 and plant cells or regenerable parts thereof, said plant parts of transgenic soybean event JK1001-1 including, but not limited to, cells, pollen, ovules, flowers, buds, roots, stems, inflorescences, leaves and products from soybean plant JK1001-1 such as soybean meal, soybean oil, soybean milk, soybean protein, soybean products and biomass left in the field of soybean crops.
The transgenic soybean event JK1001-1 of the invention comprises a DNA construct that, when expressed in a plant cell, the transgenic soybean event JK1001-1 achieves human lactoferrin gene expression and tolerance to glyphosate herbicide.
In some embodiments of the invention, the DNA construct comprises 2 expression cassettes in tandem, the first expression cassette comprising a suitable promoter for expression in a plant operably linked to a gene encoding human lactoferrin (cGmhlF) and a suitable polyadenylation signal sequence; the second expression cassette comprises a suitable promoter for expression in plants operably linked to a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (cEPSPS) and a suitable polyadenylation signal sequence, the EPSPS protein being 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 soybean endogenous gene prGMA1aB1b promoter and the soybean Tsf1 gene encoding the elongation factor EF-lalpha (prGm 17gTsf 1). The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that is functional in plants, including, but not limited to, the spattp 2 transit peptide sequence derived from arabidopsis thaliana (Arabidopsis thaliana), the terminator tNos derived from nopaline synthase, and the 3' non-transcribed sequence terminator derived from the pea nucleosome 1, 5-bisphosphate carboxylase small subunit (RbcS 2) E9 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. Such enhancers may enhance the expression level of a gene, including, but not limited to, the prGmA1aB1b enhancer and the prGm17gTsf1 enhancer. The signal peptide/transit peptide can direct the transit of the EPSPS protein to a particular organelle or compartment outside or within the cell, for example, targeting to the chloroplast using a sequence encoding a chloroplast transit peptide.
In some embodiments of the invention, a soybean cell, seed, or plant comprising transgenic soybean event JK1001-1 comprises in its genome the nucleic acid sequence of SEQ ID No. 1, SEQ ID No. 5 from positions 612 to 9071, and SEQ ID No. 2, or SEQ ID No. 5, in that order.
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 JK1001-1 with any formulation containing glyphosate herbicide will control weed growth in the field and will not affect the growth or yield of plant material derived from transgenic soybean event JK 1001-1.
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.
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 JK1001-1, whether the genomic DNA is from transgenic soybean event JK1001-1 or seed or from a plant or seed or extract of transgenic soybean event JK 1001-1. 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 fragment of SEQ ID NOS: 1-7 can be used as primers and probes for detecting soybean event JK1001-1 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 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 JK1001-1 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 JK1001-1 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 JK1001-1 of the invention, or whether a soybean sample collected from a field contains the transgenic soybean event JK1001-1, or a soybean extract, such as meal, flour, or oil, contains the transgenic soybean event JK1001-1, 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 JK 1001-1. 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 JK 1001-1. 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 JK1001-1 can be obtained by amplifying the genome of transgenic soybean event JK1001-1 using the provided primer sequences, and standard DNA sequencing of PCR amplicons 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 JK1001-1, 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 JK1001-1 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 depends 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 JK1001-1 in a sample, and can also be used to cultivate soybean plants that contain DNA from transgenic soybean event JK 1001-1. 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 JK1001-1 flanking genomic region at the 5' end of the transgene insert, a portion of the insert from the right border Region (RB) of agrobacterium, a first expression cassette consisting of the soybean endogenous gene prGmA1aB1b promoter operably linked to the human lactoferrin gene (cGmhlF) operably linked to the tNos terminator; the second expression cassette consisted of the soybean prGm17gTsf1 promoter, operably linked to the Arabidopsis chloroplast transit peptide (spatCTP 2), operably linked to the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (cEPSPS) of the Agrobacterium CP4 strain, and operably linked to the pea tPse9 terminator sequence, a portion of the insert sequence from the left border region (LB) of Agrobacterium, and the soybean plant JK1001-1 flanking genomic region (SEQ ID NO: 5) at the 3' end of the transgenic insert sequence. In the DNA amplification method, the DNA molecule used as a primer may be any part derived from the transgene insert sequence in transgenic soybean event JK1001-1 or any part derived from the DNA region of the flanking soybean genome in transgenic soybean event JK 1001-1.
Transgenic soybean event JK1001-1 can be combined with other transgenic soybean varieties, such as herbicide (e.g., 2, 4-D) tolerant soybeans. Various combinations of all of these different transgenic events, when bred with transgenic soybean event JK1001-1 of the present invention, can provide improved hybrid transgenic soybean varieties that are 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 invention provides a transgenic soybean event JK1001-1, a nucleic acid sequence for detecting a soybean plant comprising the event, and a detection method thereof, wherein the transgenic soybean event JK1001-1 contains a human lactoferrin gene and is tolerant to the phytotoxic effects of glyphosate-containing agricultural herbicides. Soybean plants of this trait express glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) proteins of human lactoferrin and agrobacterium strain CP4, which confer glyphosate tolerance to plants.
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 JK1001-1 and the detection method thereof according to the present invention;
FIG. 2 is a schematic diagram showing the structure of a recombinant expression vector JK1001 for detecting a nucleic acid sequence of a soybean plant JK1001-1 and a detection method thereof according to the present invention;
FIG. 3 is a graph of Western Blot immunoblotting results of transgenic soybean containing transgenic soybean event JK1001-1 of the present invention;
FIG. 4 is a graph of the effect of the proposed spray concentration of the transgenic soybean of the present invention comprising transgenic soybean event JK1001-1 at 4-fold dose of glyphosate herbicide.
Detailed Description
The following is a detailed description of the nucleic acid sequence and detection method of the present invention for detecting soybean plant JK 1001-1.
EXAMPLE 1 cloning and transformation
1.1 vector cloning
Recombinant expression vector JK1001 (shown in fig. 2) was constructed using standard gene cloning techniques. The vector JK1001 comprises 2 transgene expression cassettes in tandem, the first expression cassette consisting of the soybean endogenous gene prGmA1aB1b promoter operably linked to the human lactoferrin gene (cGmhlF) operably linked to the tNos terminator; the second expression cassette consisted of the soybean prGm17gTsf1 promoter operably linked to the Arabidopsis chloroplast transit peptide (spatCTP 2), to the glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (cEPSPS) of the Agrobacterium CP4 strain, and to the vegetable pea tPse9 terminator sequence. The vector JK1001 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 (variety: tianlong one) young embryos were co-cultured with Agrobacterium as described in this example 1.1 to transfer the T-DNA in the constructed recombinant expression vector JK1001 into the soybean genome to generate transgenic soybean events.
For Agrobacterium-mediated transformation of soybean, briefly, mature soybean seeds were germinated in soybean germination medium (B5 salt 3.1g/L, B5 vitamin, sucrose 20g/L, agar 8g/L, pH 5.6), seed inoculated onto germination medium, and cultured under the following conditions: the temperature is 25+/-1 ℃; photoperiod [ ]Light/dark) was 16/8h. Taking the soybean aseptic seedlings which are expanded at the cotyledonary node and are fresh green after germination for 4-6 days, cutting off hypocotyls at the position 3-4 mm below the cotyledonary node, longitudinally cutting off cotyledons, and removing terminal buds, lateral buds and seed roots. Wounds were made at cotyledonary node with the back of the scalpel, and the wounded cotyledonary node tissue was contacted with an agrobacterium suspension, wherein the agrobacterium was able to transfer the nucleotide sequence of the EPSPS gene and the nucleotide sequence of the lactoferrin gene to the wounded cotyledonary node tissue (step 1: infection step). In this step, the cotyledonary node tissue is preferably immersed in an agrobacterium suspension (OD 660 =0.5-0.8, infection medium (MS salt 2.15g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 40mg/L, 2-morpholinoethanesulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, ph 5.3) to initiate infection. The cotyledonary node tissue was co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culturing step). Preferably, the cotyledonary node tissue is cultivated after the infection step on a solid medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, 2-morpholinoethanesulfonic acid (MES) 4g/L, zeatin 2mg/L, agar 8g/L, pH 5.6). After this co-cultivation stage, there is an optional "recovery" step. In the "recovery" step, at least one antibiotic (cephalosporin 150-250 mg/L) known to inhibit the growth of Agrobacterium is present in the recovery medium (B5 salt 3.1g/L, B vitamin, 2-morpholinoethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH 5.6), and no selection agent for plant transformants is added (step 3: recovery step). Preferably, the tissue mass regenerated from cotyledonary nodes is cultured on a solid medium with antibiotics but no selection agent to eliminate agrobacterium and provide a recovery period for the infected cells. Next, the cotyledonary node regenerated tissue pieces are cultured on a medium containing a selection agent (glyphosate) and the grown transformed calli are selected (step 4: selection step). Preferably, the cotyledonary node regenerated tissue pieces are cultured on a selective solid medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholinoethanesulfonic acid (MES) 1g/L, sucrose 30g/L, 6-benzyladenine (6-BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, glyphosate isopropylamine salt 10mg/L, pH 5.6) Culturing, results in that the transformed cells can continue to grow. Then, the transformed cells are regenerated into plants (step 5: regeneration step), and preferably, the cotyledonary node regenerated tissue pieces grown on the medium containing the selection agent are cultured on solid media (B5 differentiation medium and B5 rooting medium) to regenerate the plants. The selected resistant tissue blocks are transferred to the B5 differentiation medium (B5 salt 3.1g/L, B vitamin, 2-morpholinoethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, glyphosate isopropylamine salt 10mg/L, pH 5.6) and cultured and differentiated at 25 ℃. The differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholinoethanesulfonic acid (MES) 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3-butyric acid (IBA) 1 mg/L), cultured to about 10cm high at 25℃on rooting medium, and transferred to greenhouse for cultivation until set. In the greenhouse, the cells were cultured at 26℃for 16 hours and at 20℃for 8 hours each day.
1.3 identification and screening of transgenic events
A total of 500 independent transgenic T0 individuals were generated. Molecular detection (including target gene copy number detection, insertion position analysis and the like), target character and agronomic character evaluation are carried out on the offspring of all T0 plants stably inherited, and finally JK1001-1 is obtained through screening. JK1001-1 has single copy transgene, high lactoferrin expression level, good glyphosate herbicide tolerance, and good agronomic performance.
Example 2 detection of transgenic Soybean event JK1001-1 with TaqMan
About 100mg of leaves of transgenic soybean event JK1001-1 was taken as a sample, its genomic DNA was extracted with Qiagen's DNeasyPlant Maxi Kit, and the copy numbers of epsps and cGmhlF were detected by Taqman probe fluorescent quantitative PCR method. Meanwhile, wild type soybean (transformed recipient) plants 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 JK1001-1, 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 soybean plant as a control, repeating each sample for 3 times, and taking an average value; the fluorescent quantitative PCR primer and the probe sequences are respectively as follows:
The following primers and probes were used to detect the cGmhlF gene sequence:
primer 1: TCCAATTGGCACGCTACGA, as shown in SEQ ID NO. 16 of the sequence Listing;
primer 2: ACGGCCGCCTCAATAGG, as shown in SEQ ID NO. 17 of the sequence Listing;
probe 1: CCTTCTTAAATTGGACTGGGCCTCCTGA, as shown in SEQ ID NO. 18 of the sequence Listing;
the following primers and probes were used to detect the epsps gene sequence:
primer 3: GGTGTGCAGGTGAAGTCTGAAG, as shown in SEQ ID NO 19 of the sequence Listing;
primer 4: TTGGCGTTGGAGTCTTTGGT, as shown in SEQ ID NO. 20 of the sequence Listing;
probe 2: CGGTGATCGTCTTCCAGTTACCTTGCG, as shown in SEQ ID NO. 21 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
Data were analyzed using SDS2.3 software (applied biosystems) to obtain a single copy of transgenic soybean event JK1001-1.
Example 3 transgenic soybean event JK1001-1 detection
3.1 genomic DNA extraction
DNA extraction according to the conventionally employed CTAB (cetyltrimethylammonium bromide) method: 2 g of tender transgenic soybean event JK1001-1 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 enzyme cutting is finished, 70 mu L of absolute ethyl alcohol is added into an enzyme cutting system, ice bath is carried out for 30min, centrifugal is carried out for 7min at the rotating speed of 12000rpm, the supernatant is discarded, and the enzyme cutting system is driedAdd 8.5. Mu.L of double distilled water (ddH) 2 O), 1. Mu.L of 10 XT 4 Buffer and 0.5. Mu. L T4 ligase were ligated overnight at 4 ℃. 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. 22 as a first primer, SEQ ID NO. 23, SEQ ID NO. 24 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. 25 as the first primer, SEQ ID NO. 26, SEQ ID NO. 27 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. 22. 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. 25. 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 product 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 JK 1001-1.
It was found that nucleotide 1-600 of SEQ ID NO. 5 shows the soybean genomic sequence flanking the right border (5 'flanking sequence) of the transgenic soybean event JK1001-1 insert and nucleotide 9083-9822 of SEQ ID NO. 5 shows the soybean genomic sequence flanking the left border (3' flanking sequence) of the transgenic soybean event JK1001-1 insert. 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 that is a novel DNA sequence that is diagnostic for DNA of transgenic soybean event JK1001-1 when detected in a polynucleic acid detection assay. The binding sequence of SEQ ID NO. 1 consists of 11bp on the side of the T-DNARB region insertion site and 11bp on the side of the soybean genomic DNA insertion site of transgenic soybean event JK1001-1, and the binding sequence of SEQ ID NO. 2 consists of 11bp on the side of the T-DNALB region insertion site and 11bp on the side of the soybean genomic DNA insertion site of transgenic soybean event JK 1001-1. 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 JK1001-1, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic soybean event JK1001-1 DNA. The SEQ ID NO. 6 (nucleotide 601-1170 of SEQ ID NO. 3) is a JK1001 vector DNA sequence, and the SEQ ID NO. 7 (nucleotide 1-352 of SEQ ID NO. 4) is a JK1001 vector 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 a PCR method generate a diagnostic amplicon for transgenic soybean event JK 1001-1.
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 JK 1001-1. This PCR product contains SEQ ID NO 3. For PCR amplification, primer 5 (SEQ ID NO: 8) hybridizing to the genomic DNA sequence flanking the 5' end of the transgene insert and primer 6 (SEQ ID NO: 9) located in the transgene RB sequence paired therewith were designed.
A PCR product was 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 JK 1001-1. This PCR product contains SEQ ID NO. 4. For PCR amplification, primer 8 (SEQ ID NO: 11) hybridizing to the genomic DNA sequence flanking the 3 '-end of the transgenic insert and primer 7 (SEQ ID NO: 10) of the LB sequence located at the 3' -end of the insert, paired therewith, were designed.
The DNA amplification conditions described in tables 3 and 4 can be used in the PCR zygosity assay described above to generate the diagnostic amplicon of transgenic soybean event JK 1001-1. Detection of the amplicon may be performed by using a Stratagene Robocycle, MJ Engine, perkin-Elmer 9700 or Eppendorf MastercyclerGradien 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 of transgenic soybean event JK1001-1
| Step (a) | Reagent(s) | Quantity of | Remarks |
| 1 | Water without nucleotidase | Added to the final volume 20 [ mu ] L | |
| 2 | Reaction of 10Buffer (with MgCl) 2 ） | 2.0µL | 1.5mM final buffer concentration MgCl 2 Final concentration |
| 3 | dATP, dCTP, dGTP and dTTP in 10mM | 0.4µL | 200 mu M final concentration of each dNTP |
| 4 | Event primer 5 (SEQ ID NO:8 suspended in 1X TE buffer Concentration of liquid or non-nucleotidase in water to 10 [ mu ] M | 0.2µL | Final concentration of 0.1 [ mu ] M |
| 5 | Event primer 6 (SEQ ID NO:9 suspended in 1X TE buffer Concentration of liquid or non-nucleotidase in water to 10 [ mu ] M | 0.2µL | Final concentration of 0.1 [ mu ] M |
| 6 | RNase, DNase-free (500 ng/mL) | 0.1µL | 50 ng/reaction |
| 7 | RED Taq DNA polymerase (1 unit/mu L) | 1.0 [ mu ] L (suggest in the next step) Previously converting straw | 1 unit/reaction |
| 8 | Extracted DNA (template): blade for sample to be analyzed | About 200ng | |
| Negative control | 50ng of non-transgenic soybean base Genomic DNA | ||
| Negative control | Template-free DNA (DNA re-suspension) Solutions therein | ||
| Positive control | 50ng of JK1001-1 Soybean genomic DNA |
Table 4, perkin-Elmer9700 thermal cycler conditions
| Cycle number | Setting up |
| 1 | 94 ℃ for 3 minutes |
| 34 | 94 ℃ for 30 seconds |
| 64 ℃ for 30 seconds | |
| 72 ℃ for 1 minute | |
| 1 | 72 ℃ for 10 minutes |
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 5 and 6 (SEQ ID NOS: 8 and 9), which when used in a PCR reaction of transgenic soybean event JK1001-1 genomic DNA, produced an amplification product of 1170bp fragment, and when used in a PCR reaction of non-transformed soybean genomic DNA and non-JK 1001-1 genomic DNA, NO fragment was amplified; primers 7 and 8 (SEQ ID NOS: 10 and 11), when used in the PCR reaction of transgenic soybean event JK1001-1 genomic DNA, produced an amplification product of 1092bp fragment, when used in the PCR reaction of non-transformed soybean genomic DNA and non-JK 1001-1 genomic DNA, NO fragment was amplified.
The PCR zygosity assay can also be used to identify whether the material derived from transgenic soybean event JK1001-1 is homozygous or heterozygous. Primer 9 (SEQ ID NO: 12), primer 10 (SEQ ID NO: 13) and primer 11 (SEQ ID NO: 14), or primer 10 (SEQ ID NO: 13), primer 11 (SEQ ID NO: 14) and primer 12 (SEQ ID NO: 15) are used in an amplification reaction to generate a diagnostic amplicon of transgenic soybean event JK 1001-1. 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 JK 1001-1.
TABLE 5 reaction solution for measuring the bondability
| Step (a) | Reagent(s) | Quantity of | Remarks |
| 1 | Nuclease-free water | Added to the final volume 5 [ mu ] L | |
| 2 | 2*Universal Master Mix (Applied Biosystems catalog) Number 4304437) | 5µL | Final concentration of 1 |
| 3 | Primer 9 (SEQ ID NO: 12)And primer 10 (SEQ ID NO: 13) and primer Article 11 (SEQ ID NO: 14) (resuspended in non-nucleic acid water to 10. Mu.M) Concentration of (C) | 0.3µL | Final concentration of 0.1 [ mu ] M |
| 4 | REDTaq DNA polymerase (1 unit/. Mu.L) | 1.0 [ mu ] L (suggest in the next step) Previously converting straw | 1 unit/reaction |
| 5 | Extracted DNA (template): blade for sample to be analyzed | 200ng of genomic DNA | |
| Negative control | 50ng of non-transgenic soybean base Genomic DNA | ||
| Negative control | Template-free DNA (DNA resuspension) Solution floating therein | ||
| Positive control | 50ng of JK1001-1 Soybean genomic DNA |
TABLE 6 determination of the bondability Perkin-Elmer9700 thermal cycler conditions
| Cycle number | Setting up |
| 1 | 95 ℃ for 10 minutes |
| 10 | 95 ℃ for 15 seconds |
| 64 ℃ for 1 minute (-1 ℃/cycle) | |
| 25 | 95 ℃ for 15 seconds |
| 54 ℃ for 1 minute | |
| 1 | Immersion at 10 ℃ |
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 MastercyclerGradient (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 JK1001-1 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 allele corresponding to the insert DNA present in transgenic soybean event JK 1001-1. 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 JK1001-1 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 JK1001-1 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 JK 1001-1.
The primer pair for transgenic soybean event JK1001-1 was used to generate an amplicon diagnostic for transgenic soybean event JK1001-1 genomic DNA. These primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID NOS: 8 and 9), and primers 7 and 8 (SEQ ID NOS: 10 and 11) for use in the DNA amplification method described. In addition, primers 13 and 14 (SEQ ID NO:28 and SEQ ID NO: 29) for amplifying the soybean endogenous gene are included as an intrinsic standard of the reaction conditions. Analysis of the DNA extract sample of transgenic soybean event JK1001-1 should include a positive tissue DNA extract control of transgenic soybean event JK1001-1, a negative DNA extract control derived from non-transgenic soybean event JK1001-1 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 JK1001-1, respectively, may be used. The DNA amplification conditions illustrated in tables 3-6 can be used to generate a diagnostic amplicon of transgenic soybean event JK1001-1 using an appropriate primer pair. Extracts that are presumed to contain soybean plant or seed DNA comprising transgenic soybean event JK1001-1, or products derived from transgenic soybean event JK1001-1, that when tested in a DNA amplification method produce an amplicon diagnostic for transgenic soybean event JK1001-1, can be used as templates for amplification to determine the presence or absence of transgenic soybean event JK1001-1.
Example 4 transformation event JK1001-1 lactoferrin expression Western Blot detection
The experiment selects JK1001-1 mature grains to be ground into powder for detection, the specific operation scheme is that 0.05g JK1001-1 powder is weighed, leaching is carried out for 1 hour by using 0.5 ml extracting solution (the extracting solution is prepared into 0.05M Tris, 0.5M NaCl, 0.05% TWEEN 20 and pH 7.4), centrifugation is carried out for 10 minutes at 12000 rpm, the supernatant is collected for carrying out Western Blot to carry out human lactoferrin expression detection, the detection result is shown in figure 3, wherein 1 is non-transgenic soybean, 2 is a human lactoferrin standard (the concentration is 1 mg/g), and 3 is transgenic event JK1001-1. As can be seen from fig. 3, transgenic soybean event JK1001-1 was able to successfully express human lactoferrin.
EXAMPLE 5 detection of lactoferrin content in transgenic Soybean seeds
Soybean seeds of 0.05g transgenic soybean event JK1001-1 (3 strains were set in total as 3 replicates) were taken as samples, and after grinding, 500 μl of extraction buffer (extraction buffer formulation as follows: 0.05M Tris, 0.5M NaCl, 0.05% TWEEN 20, pH 7.4) was added, and centrifuged at 12000 rpm for 10 min, the supernatant was diluted 10000 times with the extraction buffer, and 100 μl of the diluted supernatant was used for ELISA detection. The ratio of lactoferrin in the sample to fresh weight of leaf blade was detected and analyzed by ELISA (enzyme-linked immunosorbent assay) kit (Abcam company).
The measurement result of the lactoferrin content in the soybean seeds of transgenic soybean event JK1001-1 is shown in Table 7, the average lactoferrin expression amount is 2.08mg/g, and the result shows that the lactoferrin obtains higher expression amount and stability in the soybean.
TABLE 7 results of transgenic soybean event JK1001-1 lactoferrin concentration detection
Example 6 glyphosate herbicide tolerance assay for soybean transformation event
The test selects the pesticide (41% glyphosate isopropyl ammonium salt aqua) for spraying. A random block design was used, 3 replicates. Cell area of 1m 2 (1 m is multiplied by 1 m), 30 seedlings are fixed, and the conventional field experiment cultivation management is carried out. The transgenic soybean event JK1001-1 and wild soybean plants (non-transgenic, transformed recipient control (CK-) were treated by 1) spraying clear water respectively; 2) The pesticide was sprayed at a dose of 3360g a.e./ha during the V3 leaf stage and then again at the same dose during the V8 stage. 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 8. 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 ranks)/(total number×highest rank); 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 yield difference between the different treatments was measured as a yield percentage (% yield = sprayed glyphosate yield/sprayed clear water yield). The results of the tolerance of transgenic soybean event JK1001-1 to herbicides and soybean yield results are shown in fig. 4 and table 9.
TABLE 8 grading Standard of the extent of phytotoxicity of glyphosate herbicide to soybeans
| Grade of phytotoxicity | Description of symptoms |
| Level 0 | No phytotoxicity, and the growth is consistent with that of clear water contrast; |
| level 1 | Slight phytotoxicity symptoms, local color changes, and phytotoxicity spots accounting for less than 10% of the leaf area; |
| level 2 | Slightly inhibiting growth or losing green, wherein the phytotoxicity spots occupy less than 1/4 of the leaf area; |
| 3 grade | Has great influence on growth and development, and leaf malformation or plant dwarfing or phytotoxicity spots occupy less than 1/2 of the leaf area |
| Grade 4 | The influence on the growth and development is large, the serious malformation of the leaves or obvious dwarfing of the plants or less than 3/4 of the leaf spot; |
| grade 5 | The phytotoxicity is extremely serious, and the dead plants or phytotoxicity spots occupy more than 3/4 of the leaf area. |
Table 9 results of transgenic Soybean event JK1001-1 for tolerance to glyphosate herbicide and Soybean yield results
| Project/plant | JK1001-1 | CK- |
| Rate of damage (%) (spraying clear water) | 0 | 0 |
| Glyphosate rate (%) (3360 g a.e./ha nongda) | 0 | 100 |
| Yield percentage% (3360 a.e./ha nongda) | 102 | 0 |
The results show that in terms of herbicide (glyphosate) damage: 1) The transgenic soybean event JK1001-1 had a rate of victimization of substantially 0 under glyphosate herbicide (3360 g a.e./ha) treatment, whereby the transgenic soybean event JK1001-1 had good glyphosate herbicide tolerance.
In terms of yield: the yield of the transgenic soybean event JK1001-1 is not obviously different under the treatment of spraying clear water and spraying 3360g a.e./ha glyphosate 2, and after the glyphosate herbicide is sprayed, the yield of the transgenic soybean event JK1001-1 is slightly improved compared with the water spraying treatment, thereby further indicating that the transgenic soybean event JK1001-1 has good glyphosate herbicide tolerance.
In conclusion, through TaqMan TM Analysis (see example 2) of regenerated transgenic soybean plants for the presence of epsps and cGmhlF genes and characterization of stable high expression of human lactoferrin and glyphosate herbicide toleranceCopy number of sexual line. The stable expression of human lactoferrin gene was determined by Western Blot immunoblot analysis (see example 4), and event JK1001-1 was selected to be excellent by screening, having single copy transgene, highly stable expression of human lactoferrin gene (see example 5), glyphosate herbicide tolerance, and excellent agronomic performance (example 6), based on human lactoferrin gene expression level, glyphosate herbicide tolerance, and agronomic performance.
Claims (6)
1. A nucleic acid molecule for detecting a transgenic soybean event JK1001-1, wherein the sequence of the nucleic acid molecule is shown as one or more of SEQ ID NO. 1-2, SEQ ID NO. 3-4, SEQ ID NO. 5 or a complementary sequence thereof, the nucleic acid molecule is derived from the transgenic soybean event JK1001-1, and soybean seeds of the transgenic soybean event JK1001-1 have been preserved in China center for type culture collection under the preservation number CCTCC NO. P202332.
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 a DNA containing soybean event JK1001-1, an amplicon of soybean event JK1001-1 in a test sample is produced,
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 JK1001-1 are preserved in China center for type culture Collection with a preservation number CCTCC NO: P202332.
3. A method of detecting the presence of DNA of transgenic soybean event JK1001-1 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 JK 1001-1; the soybean seeds containing the soybean event JK1001-1 are preserved in China center for type culture Collection with a preservation number CCTCC NO: P202332.
4. A DNA detection kit comprising: the DNA primer pair of claim 2.
5. 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: P202332.
6. 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: P202332.
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