CN108456679B - High-oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof - Google Patents
High-oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof Download PDFInfo
- Publication number
- CN108456679B CN108456679B CN201810138900.0A CN201810138900A CN108456679B CN 108456679 B CN108456679 B CN 108456679B CN 201810138900 A CN201810138900 A CN 201810138900A CN 108456679 B CN108456679 B CN 108456679B
- Authority
- CN
- China
- Prior art keywords
- sequence
- seq
- primer
- exogenous
- site
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- 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
Abstract
The invention provides a high oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof, belonging to the technical field of plant biology. In particular to a left and right border flanking sequence of a high oleic acid transgenic soybean event E2D8037-3 exogenous insertion fragment and application thereof. The left boundary flanking sequence of the exogenous insertion fragment of the transgenic soybean event E2D8037-3 disclosed by the invention is shown as SEQ-2, and the right boundary flanking sequence is shown as SEQ-3. The flanking sequence of the exogenous insertion fragment of the transgenic soybean event E2D8037-3 disclosed by the invention can be used as a target DNA sequence to establish a specific detection method of the transgenic event. The exogenous insert flanking sequence and the detection method provided by the invention are suitable for the specific detection of the transgenic soybean event including parents, derived strains or varieties, and products thereof including plants, tissues, seeds and products.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a flanking sequence of a high-oleic acid transgenic soybean event E2D8037-3 exogenous insertion fragment and application thereof.
Background
Oleic acid is a class of unsaturated fatty acids found in the grain of a wide variety of oil crops (e.g., soybean, canola, peanut, etc.). Research shows that oleic acid has important nutritious and health functions. The oleic acid can reduce cholesterol content in blood, reduce its deposition on blood vessel, soften blood vessel, and prevent arteriosclerosis. Oleic acid is generally called as safe fatty acid in the nutrition field, and the content of the oleic acid is an important mark for evaluating the quality of the edible oil. In addition, the high proportion of oleic acid can effectively improve the stability of the edible oil due to the low degree of unsaturation of the fatty acids. The content of the oleic acid in the soybean seeds is generally 20-25%, and the proportion of the linoleic acid and the linolenic acid with higher unsaturation degree is up to more than 60%. Although linoleic acid is considered an important fatty acid and has some effect in lowering blood cholesterol levels, vegetable oils rich in linoleic acid are highly unstable and are susceptible to peroxidation when exposed to air or cooked at elevated temperatures, producing oxidation products which are harmful to health and are associated with unpleasant odours. Meanwhile, trans-fatty acid harmful to human bodies is easily generated in the processing process. In contrast, oleic acid is a monovalent unsaturated fatty acid, has high stability, and an edible oil rich in oleic acid can be used for long-term preservation and high-temperature cooking without being easily deteriorated by oxidation. In addition, the oleic acid has the nutrition and health care functions, so that the quality of the soybean high-quality grease is determined to a great extent by the content of the oleic acid. Therefore, the method has important significance for improving the soybean quality by improving the soybean oleic acid content and cultivating high-oleic-acid soybean varieties.
During seed development, the fatty acid synthase complex in the plastid catalyzes the synthesis of fatty acids from acetyl-CoA and enters the cytoplasm to synthesize triacylglycerols. OA, the precursor of LA synthesis, is the most critical step in the polyunsaturated fatty acid synthesis pathway. Delta 12-fatty acid desaturase (delta-twelve fat acid desaturase 2 enzyme, FAD2, EC 1.3.1.35) is a key enzyme that catalyzes this biochemical process, determining the content of polyunsaturated fatty acids in seeds. The existing research shows that the oleic acid/linoleic acid ratio can be obviously changed by inhibiting or over-expressing the FAD2-1 gene. Pham et al found that the oleic acid content of FAD2-1A and FAD2-1B double mutant soybeans was very significantly increased (80%). Zhang et al inhibit the expression of soybean endogenous FAD2 gene through antisense RNA mediated post-transcriptional gene silencing, and obtain transgenic soybean with oleic acid content as high as 51.71%. Wang et al obtained soybean strain with oleic acid content of 71.5% -81.9% by inhibiting soybean FAD2 gene by RNAi technology. Haun et al use artificial ribozyme Technology (TALEN) to perform site-directed mutagenesis on soybean endogenous FAD2-1A and FAD2-1B, and obtain transgenic soybean with oleic acid content of 80%.
The Jilin province agricultural science institute adopts an agrobacterium-mediated method to introduce the soybean RNAi GmFAD2-1B gene into a soybean cultivar Williams82 to obtain the high-oleic acid transgenic soybean E2D 8037-3. Continuous multi-generation fatty acid component analysis shows that the oleic acid content of the transgenic soybean E2D8037-3 reaches 77.11 percent, is obviously higher than that of a receptor control variety Williams82 (the oleic acid content is 17.81-22.0 percent), and is improved by more than 2 times. In addition, the content of the E2D8037-3 linoleic acid is reduced by about 50 percent compared with the control, the content of the saturated fatty acid, namely palmitic acid and stearic acid is reduced to about 10.0 percent, and the fatty acid composition is close to that of olive oil (the content of oleic acid is 70-80 percent). At present, the high oleic acid transgenic soybean event E2D8037-3 enters a safety evaluation stage, and with the promotion of important special items of new national transgenic organism breeding, the transformation event and derived varieties or strains thereof are expected to enter commercial application.
The specific detection of the transgenic event and the derived strain or variety thereof is an important technical means for realizing the effective supervision and management of the transgenic plant and ensuring the healthy development of the transgenic industry. The flanking sequence of the exogenous insertion fragment and the detection method established based on the flanking sequence are important basis for effective supervision and management of the transgenic plant and the product thereof. The exogenous insertion of flanking sequences into transgenic plants has been reported in related patents and literature. Zhang soldier et al (2006) analyzed the flanking sequence of the exogenous insert of maize line MON863 by TAIL-PCR method, and established a line specificity detection method of transgenic MON863 maize. Xijiajia et al (2007) obtains the flanking sequences of the exogenous insertion fragments of transgenic rice Ke-Ming-dao, Bt Shanyou 863 and Kefeng No. 6 by using TAIL-PCR, genome walking and LD-PCR and the like, Yangyou et al (2012) establishes the flanking sequences of the exogenous insertion fragments of the transgenic rice strain SK-2 by using TAIL-PCR and establishes a detection method of the specificity of the strain.
Through analysis of the existing patents and literatures, the article and patent report related to the flanking sequence of the exogenous insert of the high oleic acid transgenic soybean event E2D8037-3 are not found at present. According to the research, the left and right border flanking sequences of the exogenous insertion fragment of the transgenic soybean event E2D8037-3 are obtained by a genome re-sequencing technology and a PCR technology, and the transformation event specificity detection method is established according to the sequence characteristics of the flanking sequences, so that a basis is provided for the commercial application of the high-oleic acid transgenic soybean event E2D8037-3 and derived varieties or strains thereof. On the basis, the invention is provided.
Disclosure of Invention
The invention aims to provide high oleic acid transgenic soybean event E2D8037-3 exogenous insert left and right border flanking sequences. The invention also provides a specific detection method of the transgenic event.
The invention is realized by the following technical scheme:
the left and right border flanking sequences of the exogenous insertion fragment of the transgenic soybean event E2D8037-3 provided by the invention are shown in SEQ-2 and SEQ-3, and are characterized in that the sequence is a DNA sequence consisting of a soybean genome-derived sequence and an exogenous insertion fragment-derived sequence. Wherein:
the left border flanking sequence features of the exogenous insert include:
(1) the 1 st-213 th site sequence of SEQ-2 is derived from the genome sequence of cultivated soybean Williams 82;
(2) the 214 nd-895 th site sequence of SEQ-2 is derived from an exogenous insertion fragment sequence.
The characteristics of the right border flanking sequence of the exogenous insert include:
(1) the 1 st-615 th site sequence of SEQ-3 comes from the exogenous insertion fragment sequence;
(2) the 616 nd 998 th site sequence of SEQ-3 is derived from the genome sequence of cultivated soybean Williams 82.
The flanking sequences of the left border and the right border of the exogenous insertion fragment of the transgenic soybean event E2D8037-3 provided by the invention are obtained by the following steps: (1) taking a high oleic acid transgenic soybean event E2D8037-3 as a material, adopting a genome re-sequencing technology, searching a soybean genome database (http:// soybase. org /), determining that the specific insertion position of an exogenous fragment in a reference soybean genome (Wm82.a2.v1) is 45341084 site of Chr15 chromosome, and the insertion mode is single-site double-copy insertion. And obtaining a sequence of 2kb of the insertion site at the upstream and downstream of the reference soybean genome, as shown in SEQ-1. (2) Primers are designed according to the upstream and downstream sequences of the exogenous fragment and the sequence of the insert fragment in a reference soybean genome, and PCR amplification is carried out by taking the total DNA of the E2D8037-3 genome as a template to obtain the flanking sequences of the left and right boundaries of the exogenous insert fragment of the transgenic soybean event E2D8037-3, wherein the flanking sequences are shown as SEQ-2 and SEQ-3. The sequence is composed of DNA sequence from soybean genome sequence and exogenous insertion fragment sequence.
In view of the random integration of the exogenous fragment in the plant genome in the transgenic event, the insertion sites of the exogenous fragment in the genome are different in different transgenic events. The flanking sequences are specific for a particular transgenic event. Thus, the use of flanking sequences of the insert allows for the specific detection of the transgenic event. For example, hybridization is performed using a probe containing a part of the flanking sequence and a part of the foreign insert sequence, or PCR amplification is performed by designing a specific primer containing a part of the flanking sequence and a part of the foreign insert sequence.
The specific detection method or detection kit for the transgenic soybean event E2D8037-3 is characterized in that a specific detection primer is designed or a specific probe is prepared by utilizing the left boundary flanking sequence of the exogenous insertion segment of the transgenic soybean event E2D 8037-3. Wherein one primer is a forward primer designed according to the sequence at the 1 st-213 th site of SEQ-2, and the other primer is a reverse primer designed according to the sequence at the 214 nd-895 th site of SEQ-2, i.e., the two primer combinations are the primers for detecting the specificity of the flanking sequence at the left boundary of the exogenous insert as described in claim 1.
Preferably, the detection primer specific to the left border flanking sequence of the exogenous insert is:
the forward primer is as follows: 5'-CCACCACTCCCAATGACAACA-3' (SEQ-4)
The reverse primer is as follows: 5'-GCATCTTGAACGATAGCCTTTCCT-3' (SEQ-5)
The specific detection method or detection kit for the transgenic soybean event E2D8037-3 is characterized in that a specific detection primer is designed or a specific probe is prepared by utilizing the right boundary flanking sequence of the exogenous insertion fragment of the transgenic soybean event E2D 8037-3. One primer is a forward primer designed according to the sequence at the 1 st-615 th site of SEQ-3, and the other primer is a reverse primer designed according to the sequence at the 616 nd 998 th site of SEQ-3, i.e., the two primer combinations are the primers for detecting the specificity of the flanking sequence at the right boundary of the exogenous insert fragment as described in claim 1.
Preferably, the detection primer specific to the right border flanking sequence of the exogenous insert is:
the forward primer is as follows: 5'-GGGAGAGGCGGTTTGCGTATT-3' (SEQ-6)
The reverse primer is as follows: 5'-GAGGGATAGTAACTCATGCAATAAACG-3' (SEQ-7)
The invention provides application of flanking sequences of a left boundary and a right boundary of an exogenous insertion segment of a transgenic soybean event E2D8037-3 and a specificity detection method in detection of transgenic soybean event E2D8037-3 including parents, derived strains or varieties, and products thereof including plants, tissues, seeds and products. Designing specific detection primers as shown in SEQ-4 and SEQ-5 according to the left boundary flanking sequence of the E2D8037-3 exogenous insert; or designing specific detection primers according to the flanking sequence of the right boundary of the E2D8037-3 exogenous insert, wherein the specific detection primers are shown as SEQ-6 and SEQ-7. Transgenic soybean event E2D8037-3 root, stem, leaf, flower and seed DNA samples were extracted separately and PCR amplification was performed with recipient non-transgenic soybean variety Williams82 and a conventional soybean variety as controls. The PCR products were separated by electrophoresis on a 1% agarose gel and stained with EB to identify the presence of specifically amplified bands. The length of the left border amplification fragment of the transgenic soybean event E2D8037-3 exogenous insertion fragment is 438 bp. The length of the right border amplification fragment of the transgenic soybean event E2D8037-3 exogenous insertion fragment is 332 bp.
In the invention, transgenic soybean event E2D8037-3 is obtained by introducing a soybean GmFAD2-1B gene RNAi fragment into a soybean cultivar Williams82 by an agrobacterium-mediated method. The structural map of the E2D8037-3 transformation vector pTF101-BCSP-GmFAD2-1Bi is shown in figure 1. The vector carries an RNAi fragment expression cassette of a GmFAD2-1B gene and a screening marker gene BAR expression cassette. The transformation vector pTF101-BCSP-GmFAD2-1Bi and transgenic soybean event E2D8037-3 are publicly available from the agricultural scientific college of Jilin province.
In the invention, the specific detection method of the transgenic soybean event E2D8037-3 comprises the following steps of: 2.5uL of 10 XPCR buffer, 0.5uL of 10mmol/L dNTPs, 0.5uL of 5U/uL Taq enzyme, 1.0uL of DNA sample, 0.5uL of 10umol/L forward primer, 0.5uL of 10umol/L reverse primer, and ddH2O19.5 uL. The PCR reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; 5min at 72 ℃. Detecting whether a specific band exists in the PCR amplification product by using 1% agarose gel electrophoresis, and analyzing whether a sample contains a component derived from E2D 8037-3.
The invention has the advantages of
1. The invention discloses flanking sequences of a left boundary and a right boundary of a high oleic acid transgenic soybean event E2D8037-3 exogenous insert for the first time.
2. The invention firstly analyzes and confirms the composition of the left and right boundary flanking sequences of the high oleic acid transgenic soybean event E2D8037-3 exogenous insertion fragment, including the sequence of the exogenous insertion fragment and the genome sequence of cultivated soybean Williams82, and determines the specific insertion site of the exogenous fragment in the soybean genome.
3. By utilizing the characteristics of the flanking sequences of the left boundary and the right boundary of the exogenous insert provided by the invention, a high oleic acid transgenic soybean event E2D8037-3 specific qualitative PCR detection method or a detection kit is established.
4. By utilizing the exogenous insert left and right border flanking sequences and the specificity detection method provided by the invention, the specificity detection is carried out on the transgenic soybean event E2D8037-3 including parents, derived strains or varieties and products thereof including plants, tissues, seeds and products, thereby realizing the effective supervision and management of the transgenic soybean and the products thereof.
Description of the drawings:
FIG. 1 shows a structural map of E2D8037-3 transformation vector pTF101-BCSP-GmFAD2-1Bi
Figure 2. transgenic soybean event E2D8037-3 left border flanking sequence specific PCR detection. M: DNA molecular weight standard (DL2000), 1: E2D8037-3, 2: E2D8037-3 stem, 3: E2D8037-3 leaf, 4: E2D8037-3 flower, 5: E2D8037-3 seed, 6: soybean variety Williams82, 7: soybean variety Jiyu 47, 8: soybean variety Jiyu 72, 9: corn, 10: cotton
Figure 3 transgenic soybean event E2D8037-3 right border flanking sequence specific PCR detection.m: DNA molecular weight standard (DL2000), 1: E2D8037-3, 2: E2D8037-3 stem, 3: E2D8037-3 leaf, 4: E2D8037-3 flower, 5: E2D8037-3 seed, 6: soybean variety Williams82, 7: soybean variety Jiyu 47, 8: soybean variety Jiyu 72, 9: corn, 10: cotton
Detailed Description
The present invention will be further described with reference to specific embodiments for the purpose of facilitating an understanding of technical means, characteristics of creation, objectives and functions realized by the present invention, but the following embodiments are only preferred embodiments of the present invention, and are not intended to be exhaustive. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 transgenic Soybean event E2D8037-3 exogenous fragment insertion site analysis
1. Transgenic soybean E2D8037-3 genome DNA extraction
(1) Extracting genome DNA: taking 1-2g of soybean young leaves, grinding the soybean young leaves into powder by using liquid nitrogen, and filling the powder into a 50mL centrifuge tube. 5mL of extract A (100mmol/L Tris-HCl, pH8.0, 0.35mol/L sorbitol, 5mmol/L EDTA, pH8.0, 1% 2-mercaptoethanol), 3.5mL of extract B (50mmol/L Tris-HCl, pH8.0, 4.0mol/L NaCl, 1.8% CTAB, 25mmol/L EDTA, pH8.0), 0.3mL 30% sodium lauroyl sarcosinate and 2% PVP-360 were added in this order, and incubated at 55 ℃ for 60 to 90 minutes while shaking gently several times. The tube was removed, added with chloroform/isoamyl alcohol (24: 1) of the same volume, shaken gently upside down for 15 minutes, and then centrifuged at room temperature for 10 minutes (13000 rpm). The supernatant was aspirated, 2/3 volumes of pre-cooled isopropanol mixed with 1/10 volumes of sodium acetate in the supernatant were added, and centrifuged at 13000rpm for 20 minutes at 4 ℃. The supernatant was discarded and rinsed with cold 75% ethanol. Air drying the DNA to surface, and storing at-20 deg.C
(2) And (3) genomic DNA purification: with 200uL ddH2O-solubilized DNA, 5uL RNase (10mg/mL) was added, and incubated at 37 ℃ for 40 minutes. Extracted 1-2 times with equal volume of phenol/chloroform and centrifuged at 13000rpm for 10 minutes at room temperature. The supernatant was transferred to a new 1.5mL centrifuge tube and precipitated with an equal volume of pre-cooled 100% chloroform. Centrifuge at 13000rpm for 10 minutes at room temperature. The supernatant was transferred to a new 2mL centrifuge tube and the DNA was precipitated with an amphiploid volume of cold absolute ethanol (1/10 volumes of sodium acetate mixed) and then left at-20 ℃ for 30 minutes. 13000rpm for 15 minutes, rinsing with 75% ethanol for 2 times, and drying in air for 15-20 minutes. 50-100 uL ddH2O dissolves the DNA. After the DNA concentration was measured by an ultraviolet spectrophotometer (Quawell Q5000), it was stored at-20 ℃ until use.
2. Transgenic soybean E2D8037-3 genome re-sequencing analysis
The transgenic soybean E2D8037-3 was subjected to re-sequencing analysis by Beijing Baimaike Biotech Co. Fragmenting qualified sample genome DNA by using ultrasonic waves, and then purifying, repairing the tail end, adding A to the 3' end and connecting a sequencing joint to the fragmented DNA. And then agarose gel electrophoresis is carried out to select the size of the fragment, and PCR amplification is carried out to form a sequencing library. Sequencing qualified libraries by using a second-generation high-throughput sequencing Xten platform. The sequenced 64079515 original Reads (paired sequences) were quality assessed and filtered to yield 62715961 clear Reads. Clear Reads were then aligned to the reference genomic sequence (wm82.a2.v1, http:// phytozome. jgi. doe. gov/pz/portal. html # | infoalias ═ Org _ Gmax). And (3) positioning the positions of the Clean Reads on the reference genome through alignment, and counting information such as sequencing depth, genome coverage and the like of each sample. The Data volume of this analysis was 19.20Gbp of Clean Data, with Q30 reaching 85.05%. The average alignment of the sample to the reference genome was 99.48%, the average depth of coverage was 17X, and the genome coverage was 98.82% (at least one base coverage).
Comparing the re-sequencing data of the transgenic soybean E2D8037-3 with a reference genome and an exogenous insertion sequence respectively, and finding out the following two types of Paired _ end reads according to the comparison result: the first type is that a reference genome sequence is aligned on a reads at one end, and an insertion sequence is aligned on a reads at the other end; the second type is that a part of reads at either end is aligned with the reference genome sequence, and the other part is aligned with the insert sequence. Bwa is used to align the reference genome, and all reads that align with the exogenous insertion are selected for local assembly. And respectively comparing the exogenous insertion sequence with the reference genome result by using blastn according to the assembled contig, selecting regions of the contig sequence compared to the chromosome, and carrying out IGV screenshot verification on bwa comparison results of the regions to obtain the insertion position information of the exogenous insertion fragment. Analysis results show that the insertion position of the exogenous fragment of the transgenic soybean E2D8037-3 is the 45341084 site of the chromosome of Chr15, and the insertion mode is single-site double-copy insertion. The position of the insertion site in the reference genomic sequence (Wm82.a2.v1) and the-2 kb sequences upstream and downstream thereof (Gm 15: 45340084..45342083) are shown in SEQ-1.
Example 2 analysis of the left and right border flanking sequences of the exogenous insert of transgenic Soybean event E2D8037-3
And designing PCR detection primers according to the exogenous insertion sequence of the transgenic soybean event E2D8037-3 and upstream and downstream sequences of the insertion site in the soybean reference genome. The E2D8037-3 insertion site upstream sequence amplification primer is E2D8037 LB-F1: (5'-CGGAAGTTGACCGTGCTTGT-3') and E2D8037LB-R1 (5'-GCCACCACTCCCAATGACAA-3'); the downstream sequence amplification primers of the E2D8037-3 insertion site are E2D8037RB-F1 (5'-CGGTAGAGCAAGTAAGTCACAG-3') and E2D8037RB-R1 (5'-TGTTGTCATTGGGAGTGGTGG-3').
The E2D8037-3 genomic DNA is used as a template, and the primers are used for PCR amplification. The PCR reaction system (25uL) was: 2.5uL of 10 XPCR buffer, 0.5uL of 10mmol/L dNTPs, 0.5uL of 5U/uL Taq enzyme, 1.0uL of sample DNA, 0.5uL of 10umol/L forward primer, 0.5uL of 10umol/L reverse primer, and ddH2O19.5 uL. The PCR reaction conditions are as follows: 5min at 95 ℃; at 94 ℃ for 45s, at 60 ℃ for 45s and at 72 ℃ for 3min, for 35 cycles; 72 ℃ for 15 min. The PCR amplification product was detected by electrophoresis on a 1% agarose gel. The PCR product was then purified using a gel recovery kit and ligated into the EZ-T cloning vector from GENSTAR. And (3) trusting Shanghai workers to carry out sequencing verification, and comparing a sequencing result with the exogenous insertion sequence and the reference genome sequence to finally obtain a transgenic soybean event E2D8037-3 exogenous insertion fragment, wherein the left boundary flanking sequence is shown as SEQ-2, and the right boundary flanking sequence is shown as SEQ-3. The sequence consists of a genomic sequence derived from cultivated soybean Williams82 and a DNA sequence derived from a foreign insert sequence.
The left border flanking sequence (SEQ-2) of the E2D8037-3 exogenous insert is 895bp in length, wherein the site sequence 1-213 is derived from the genome sequence of cultivated soybean Williams82, and the site sequence 214-895 is derived from the exogenous insert sequence. The E2D8037-3 exogenous insert right border flanking sequence (SEQ-3) is 998bp in length, wherein the site sequence 1-615 is derived from the exogenous insert sequence, and the site sequence 616-998 is derived from the genome sequence of the cultivated soybean Williams 82.
Example 3 transgenic Soybean event E2D8037-3 specific PCR detection
Specific detection primers are respectively designed according to a left boundary flanking sequence (shown as SEQ-2) and a right boundary flanking sequence (shown as SEQ-3) of the exogenous insert of the transgenic soybean event E2D 8037-3. In the left boundary flanking sequence specificity detection primer combination, one primer is a forward primer designed according to the 1 st to 213 th site sequence of SEQ-2 and is shown as SEQ-4; the other primer is a reverse primer designed according to the 214-895 th site sequence of SEQ-2, and is shown as SEQ-5. In the primer combination for detecting the specificity of the flanking sequences on the right boundary, one primer is a forward primer designed according to the sequence of the 1 st to 615 th sites of SEQ-3, and is shown as SEQ-6; the other primer is a reverse primer designed according to the 616 th and 998 th site sequences of SEQ-3 and is shown as SEQ-7.
DNA samples of roots, stems, leaves, flowers and seeds of transgenic soybean plant E2D8037-3 were extracted, respectively, according to the method of example 1. PCR amplification was performed using recipient non-transgenic soybean variety Williams82, conventional soybean variety Jiyu 47, Jiyu 72, and corn and cotton as controls. The PCR reaction system (25uL) was: 2.5uL of 10 XPCR buffer, 0.5uL of 10mmol/L dNTPs, 0.5uL of 5U/uL Taq enzyme, 1.0uL of DNA sample, 0.5uL of 10umol/L forward primer, 0.5uL of 10umol/L reverse primer, and ddH2O19.5uL. The PCR reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; 5min at 72 ℃. The PCR products were separated by electrophoresis on a 1% agarose gel and stained with EB to identify the presence of specifically amplified bands. When the specific primers are used for PCR amplification, amplification bands of a non-transgenic soybean variety Williams82, a conventional soybean variety Jiyu 47 and Jiyu 72 and corn and cotton are not generated, only a transgenic soybean E2D8037-3 sample comprises roots, stems, leaves, flowers and seeds to generate a specific amplification band, wherein the length of an amplification fragment of a left boundary flanking sequence is 438bp, and is shown in figure 2; the amplified fragment of the right border flanking sequence was 332bp in length, as shown in FIG. 3. The research shows that PCR analysis is carried out by using the exogenous insert flanking sequence specific primer, and whether the sample contains the component from E2D8037-3 can be specifically detected.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. The high oleic acid transgenic soybean event E2D8037-3 exogenous insert left border and right border flanking sequences are characterized in that the exogenous insert left border flanking sequence is shown as SEQ-2, the exogenous insert right border flanking sequence is shown as SEQ-3, the exogenous insert left border and right border flanking sequences consist of a soybean genome-derived sequence and an exogenous insert-derived sequence,
the left border flanking sequence of the foreign insert comprises:
(1) the 1 st-213 th site sequence of SEQ-2 is derived from the genome sequence of cultivated soybean Williams 82;
(2) the 214-895 site sequence of SEQ-2 is derived from an exogenous insertion fragment sequence;
the right border flanking sequence of the foreign insert comprises:
(1) the 1 st-615 th site sequence of SEQ-3 comes from the exogenous insertion fragment sequence;
(2) the 616 nd 998 th site sequence of SEQ-3 is derived from the genome sequence of cultivated soybean Williams 82.
2. The method for preparing the flanking sequences of the left and right borders of the exogenous insertion fragment as claimed in claim 1, wherein the exogenous insertion fragment is obtained by extracting the genomic DNA of the transgenic soybean event E2D8037-3, analyzing and determining the insertion site of the exogenous fragment by using a genome re-sequencing technology and amplifying by PCR.
3. Use of a sequence according to claim 1 for establishing a specific detection method or for preparing a detection kit for transgenic soybean event E2D8037-3, characterized in that specific primers or probes are prepared based on the sequence according to claim 1.
4. Use of the sequence of claim 1, the specific detection method or the detection kit of claim 3 in the detection of transgenic soybean event E2D8037-3, wherein the object of detection comprises a parent, a derived line or variety, and a product or product thereof.
5. The specific detection method as set forth in claim 3 or 4, wherein a combination of two primers is used, wherein one primer is a forward primer designed based on the sequence at the 1 st to 213 th sites of SEQ-2, and the other primer is a reverse primer designed based on the sequence at the 214 nd and 895 th sites of SEQ-2, i.e., the combination of the two primers is the specific detection primer for the left border flanking sequence of the exogenous insertion fragment as set forth in claim 1.
6. The detection kit as claimed in claim 3 or 4, wherein a combination of two primers is used, wherein one primer is a forward primer designed according to the sequence at the 1 st-213 th site of SEQ-2, and the other primer is a reverse primer designed according to the sequence at the 214 nd-895 th site of SEQ-2, i.e., the combination of the two primers is the detection primer specific to the left border flanking sequence of the exogenous insertion fragment as claimed in claim 1.
7. The primer for detecting the specificity of the flanking sequence of the left border of the exogenous insertion fragment according to claim 5, which is characterized by comprising the sequences shown in SEQ-4 and SEQ-5:
the forward primer SEQ-4 is: 5'-CCACCACTCCCAATGACAACA-3'
The reverse primer SEQ-5 is: 5'-GCATCTTGAACGATAGCCTTTCCT-3' are provided.
8. The specific detection method as claimed in claim 3 or 4, wherein a combination of two primers is used, wherein one primer is a forward primer designed according to the sequence at the 1 st-615 th site of SEQ-3, and the other primer is a reverse primer designed according to the sequence at the 616 nd-998 th site of SEQ-3, i.e., the combination of the two primers is the specific detection primer for the right border flanking sequence of the exogenous insertion fragment as claimed in claim 1.
9. The detection kit as claimed in claim 3 or 4, wherein a combination of two primers is used, wherein one primer is a forward primer designed according to the sequence at the 1 st-615 th site of SEQ-3, and the other primer is a reverse primer designed according to the sequence at the 616 nd-998 th site of SEQ-3, i.e., the combination of the two primers is the detection primer specific to the right border flanking sequence of the exogenous insertion fragment as claimed in claim 1.
10. The primer for detecting the specificity of the right border flanking sequence of the exogenous insertion fragment according to claim 7, wherein the primer set forth in SEQ-6 and SEQ-7:
the forward primer SEQ-6 is: 5'-GGGAGAGGCGGTTTGCGTATT-3'
The reverse primer SEQ-7 is: 5'-GAGGGATAGTAACTCATGCAATAAACG-3' are provided.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810138900.0A CN108456679B (en) | 2018-02-03 | 2018-02-03 | High-oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810138900.0A CN108456679B (en) | 2018-02-03 | 2018-02-03 | High-oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108456679A CN108456679A (en) | 2018-08-28 |
| CN108456679B true CN108456679B (en) | 2021-07-30 |
Family
ID=63240033
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810138900.0A Expired - Fee Related CN108456679B (en) | 2018-02-03 | 2018-02-03 | High-oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108456679B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904268A (en) * | 2019-12-30 | 2020-03-24 | 昆明理工大学 | Probe primer combination, kit and method for detecting high-oleic acid transgenic soybean |
| CN113528512B (en) * | 2021-08-06 | 2022-04-08 | 中国农业科学院油料作物研究所 | Flanking sequence of soybean GmWRI1b-OX6 with transferred GmWRI1b gene, primer pair for detection and detection method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724669A (en) * | 2005-03-17 | 2006-01-25 | 东北师范大学 | Method in increasing oleic acid content of soybean and peanut seed by applying gene silent technology |
| CN101421406A (en) * | 2006-02-13 | 2009-04-29 | 孟山都技术有限公司 | Nucleic acid constructs and methods for producing altered seed oil compositions |
| CN102127540A (en) * | 2010-12-22 | 2011-07-20 | 中国科学院植物研究所 | Method for improving soybean quality and DNA (Deoxyribonucleic Acid) molecule special for same |
| CN102164476A (en) * | 2008-09-29 | 2011-08-24 | 孟山都技术公司 | Soybean transgenic event MON87705 and methods for detection thereof |
| CN105567682A (en) * | 2016-01-12 | 2016-05-11 | 吉林省农业科学院 | Transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof |
-
2018
- 2018-02-03 CN CN201810138900.0A patent/CN108456679B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724669A (en) * | 2005-03-17 | 2006-01-25 | 东北师范大学 | Method in increasing oleic acid content of soybean and peanut seed by applying gene silent technology |
| CN101421406A (en) * | 2006-02-13 | 2009-04-29 | 孟山都技术有限公司 | Nucleic acid constructs and methods for producing altered seed oil compositions |
| CN102164476A (en) * | 2008-09-29 | 2011-08-24 | 孟山都技术公司 | Soybean transgenic event MON87705 and methods for detection thereof |
| CN102127540A (en) * | 2010-12-22 | 2011-07-20 | 中国科学院植物研究所 | Method for improving soybean quality and DNA (Deoxyribonucleic Acid) molecule special for same |
| CN105567682A (en) * | 2016-01-12 | 2016-05-11 | 吉林省农业科学院 | Transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof |
Non-Patent Citations (3)
| Title |
|---|
| An Intron Sense Suppression Construct Targeting Soybean FAD2-1 Requires a Double-Stranded RNA-Producing Inverted Repeat T-DNA Insert;Andrew Mroczka等;《Plant Physiology》;20100630;第153卷;第882-891页 * |
| 反义RNA 介导GmFAD2-1B基因沉默增强大豆种子中油酸的高效累积;杨静等;《作物学报》;20171231;第43卷(第11期);第1588-1595页 * |
| 基于基因组重测序的高含量油酸转基因大豆T-DNA旁侧序列分析及事件特异性PCR检测;仲晓芳等;《农业生物技术学报》;20181231;第26卷(第12期);第2017-2026页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108456679A (en) | 2018-08-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kasai et al. | Graft-transmitted siRNA signal from the root induces visual manifestation of endogenous post-transcriptional gene silencing in the scion | |
| CN108430213A (en) | Definite inflorescence castor-oil plant | |
| CN108456679B (en) | High-oleic acid transgenic soybean event E2D8037-3 exogenous insert flanking sequence and application thereof | |
| CN111500763A (en) | SNP molecular marker related to palmitoleic acid content in oil tea seed oil and application thereof | |
| Gomez et al. | CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production | |
| CN108660140B (en) | Application of SlSL4 gene in regulation and control of tomato fruit ripening | |
| CN108179146B (en) | Disease-resistant transgenic soybean event B5C9120-3 exogenous insert flanking sequence and application thereof | |
| CN108239640B (en) | Disease-resistant transgenic soybean event B5C9122-2 exogenous insert flanking sequence and application thereof | |
| CN108179147B (en) | High-oleic acid transgenic soybean event E2D9050 exogenous insert flanking sequence and application thereof | |
| CN108374007B (en) | High-oleic acid transgenic soybean event EB8072 exogenous insert flanking sequence and application thereof | |
| WO2021195780A1 (en) | Methods of determining sensitivity to photoperiod in cannabis | |
| CN110331223B (en) | Molecular marker, primer pair, kit and method for identifying different cane shoots types | |
| CN102604963B (en) | Separation, cloning and application of Poncirus trifoliata EARLYFLOWERING 5 (PtELF5) gene | |
| CN108374006B (en) | Disease-resistant transgenic soybean event B5C9123-5 exogenous insert flanking sequence and application thereof | |
| CN108239675B (en) | Molecular marker TJcM02 for identifying melon unisexual flower and application thereof | |
| CN108531636B (en) | Molecular marker TJcM01 for identifying melon unisexual flower and application thereof | |
| Wei et al. | In planta genetic transformation to produce CRISPRed high-oleic peanut | |
| CN109988861B (en) | Flanking sequence for detecting high oleic acid transgenic soybean E2D5022 and detection method thereof | |
| CN108239639B (en) | Flanking sequence of exogenous insert of stress-tolerant transgenic soybean event WB1 and application thereof | |
| CN104293808B (en) | A kind of hybridized Chinese tuliptree LhMKK2 genes and its expressing protein and application | |
| CN108239637B (en) | Disease-resistant transgenic soybean event B5B8127-3 exogenous insert flanking sequence and application thereof | |
| CA2835170A1 (en) | Development of phytophthora resistant potato with increased yield | |
| CN108239641B (en) | Disease-resistant transgenic soybean event B5B9104-3 exogenous insert flanking sequence and application thereof | |
| CN112813181A (en) | Reference gene suitable for gene expression analysis of different tissues of cigar and application thereof | |
| CN109897913B (en) | Flanking sequence for detecting hrf2 gene-transferred disease-resistant soybean B1A9130 and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210730 Termination date: 20220203 |