CN112410461B - Anti-mosaic virus transgenic soybean event SMV-2 exogenous insert fragment flanking sequence and application thereof - Google Patents

Abstract

The invention discloses a mosaic virus resistant transgenic soybean event SMV-2 exogenous insert fragment flanking, which comprises a left boundary flanking and a right boundary flanking. The invention also provides application of the exogenous insert fragment flanking of the mosaic virus resistant transgenic soybean event SMV-2: and detecting whether the sample to be detected contains the component derived from SMV-2. The sample to be tested comprises a plant, a tissue, a seed and a product corresponding to the parent, the derivative strain or the variety of the SMV-2.

Description

Anti-mosaic virus transgenic soybean event SMV-2 exogenous insert fragment flanking sequence and application thereof

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to left and right boundary flanking sequences of an anti-mosaic virus transgenic soybean event SMV-2 exogenous insert fragment and application thereof.

Background

The detection of transgenic plant specific transformation event is carried out by designing corresponding primers according to known flanking sequences depending on the flanking sequence analysis of the insertion site of the exogenous gene. Therefore, it is important to specify information on the T-DNA insertion site for successful development of the related studies. In transgenic production applications, the insertion site of the foreign gene is a tag of each gene transformation event, and screening and identification of transformation events, safety evaluation of transformation events, environmental release application and the like are all required to provide a T-DNA insertion site and flanking sequences of the inserted gene for each gene transformation event.

The current methods for obtaining the flanking sequences of the T-DNA insertion site are divided into 3 classes according to the principle: inverse PCR, exogenous linker-mediated PCR, semi-random primer PCR (e.g., tail-PCR). In addition, other methods such as plasmid rescue, PCR-walking, etc., have been widely used for identification of insertion sites and acquisition of flanking sequences. However, these methods have the disadvantages of complicated operation steps, long time consumption, and the like, and have large uncertainty, poor specificity, and cannot obtain high success rate.

In recent years, with the continuous development of sequencing technology, whole genome resequencing technology has become mature. The time and cost of whole genome re-sequencing are greatly reduced, so that the whole genome re-sequencing is more and more widely applied, and the whole genome re-sequencing is used for measuring the whole genome sequences of different species, constructing a whole genome map, cloning mutant genes, analyzing sequences beside insertion sites and the like.

Transgenic soybean event SMV-2 is obtained by introducing an exogenous SMV-CP gene RNAi fragment into Tianlong No. one of a cultivated soybean variety by using an Agrobacterium-mediated method. The SMV-2 transformation vector is RNAi expression vector p7 GWIGG 2-CP, and the structural diagram is shown in figure 1. The RNAi expression vector contains a 616bp conserved region fragment of a CP gene interfering with a Wuhan SMV strain G7 (AY 216010), contains a BAR gene which codes for phosphinothricin acetyltransferase, and is used as a screening marker and a target gene to show resistance to herbicide glufosinate, and the used agrobacterium strain is EHA105. The transformation vector p7 GWIGG 2-CP and transgenic soybean event SMV-2 are available from university of Zhejiang crop science research. The agrobacterium-mediated transformation of the RNAiCP gene in soybean has been published in volume 39, 9, 1594-1601, crop journal 2013.

Disclosure of Invention

The invention aims to solve the technical problem of providing a mosaic virus disease-resistant transgenic soybean event SMV-2 exogenous insert fragment flanking sequence and application thereof.

In order to solve the technical problems, the invention provides a mosaic virus-resistant transgenic soybean event SMV-2 exogenous insert fragment flanking: 500bp flanking the left border is shown in SEQ ID NO. 1; 500bp flanking the right border is shown in SEQ ID NO. 2.

Improvement of the flanking of the exogenous insert for the anti-mosaic virus transgenic soybean event SMV-2 as the invention: a DNA sequence consisting of a soybean-derived genomic sequence and an exogenous insert sequence; as described in SEQ ID NO. 5;

the SEQ ID NO. 5 is 6513bp, and the 1 st-500 th site sequence of the SEQ ID NO. 5 is derived from a genome sequence (SEQ ID NO. 1 sequence, namely a left flanking sequence of exogenous T-DNA insertion) of the cultivated soybean Tianlong No. 1; the sequence of the 501 th to 6013 th sites of SEQ ID NO. 5 is derived from an exogenous insert sequence; the sequence at positions 6014-6513 of SEQ ID NO. 5 is derived from the genomic sequence of Tianlong-number-one of cultivated soybean (SEQ ID NO. 2, the right flanking sequence of the foreign T-DNA insertion).

The invention also provides a preparation method of the mosaic virus resistant transgenic soybean event SMV-2 exogenous insert fragment flanking: the transgenic soybean event SMV-2 genomic DNA is extracted, the exogenous fragment insertion site is determined by utilizing the analysis of a genome re-sequencing technology, and the exogenous fragment insertion site is obtained by PCR amplification.

The invention also provides application of the exogenous insert fragment flanking of the mosaic virus resistant transgenic soybean event SMV-2: and detecting whether the sample to be detected contains the component derived from SMV-2.

As an improvement of the use of the invention:

the SMV-2 left boundary detection primer is:

the forward primer is as follows: LB-F1, 5'-TAATCCATTTTGCCTCGC-3', the total number of the components,

the reverse primer is as follows: LB-R1:5'-TCAATTCGGCGTTAATTCAG-3';

the expected amplified fragment size was 831bp.

That is, when the 831bp fragment was obtained by amplification, it was judged that the sample to be tested contained a component derived from SMV-2. Otherwise, it is not contained.

As an improvement of the use of the invention:

the SMV-2 right border detection primer is:

the forward primer is as follows: 5'-CGTTTCCCGCCTTCAGTT-3' the number of the individual pieces of the plastic,

the reverse primer is as follows: 5'-TGGCGGAATCACCGTAACC-3';

the expected amplified fragment size is 668bp.

That is, when the 668bp fragment was obtained by amplification, it was judged that the sample to be tested contained a component derived from SMV-2. Otherwise, it is not contained.

Either the SMV-2 left border detection primer or the SMV-2 right border detection primer may be selected and judged based on the results. The two are performed simultaneously, so that the detection result can be ensured to be more accurate.

As a further improvement of the use according to the invention:

the sample to be tested comprises a plant, a tissue, a seed and a product corresponding to a parent, a derivative strain or a variety of SMV-2.

In the present invention:

specific detection primers designed based on the SMV-2 exogenous insert left border flanking sequence (SMV-2 left border detection primers): one of the primers is a forward primer designed according to the sequence of the 1 st to 500 th sites of SEQ ID NO. 5, the other primer is a reverse primer designed according to the sequence of the 501 st to 833 th sites of SEQ ID NO. 5,

specific detection primers (SMV-2 right border detection primers) designed based on the SMV-2 exogenous insert right border flanking sequences: one of the primers is a forward primer designed according to the sequence of the 5814-6013 locus of SEQ ID NO. 5, and the other primer is a reverse primer designed according to the sequence of the 6014-6513 locus of SEQ ID NO. 5.

The invention respectively extracts DNA samples of the T5 generation transgenic soybean event SMV-2 roots, stems, leaves, flowers and seeds, and uses the acceptor non-transgenic soybean variety Tianlong No. I as a control for PCR amplification. The PCR products were separated by 1% agarose gel electrophoresis and stained with EB to identify the presence of specific amplified bands. The left boundary amplified fragment length of the exogenous insert of the transgenic soybean event SMV-2 is 831bp (FIG. 2 and FIG. 4), and the right boundary amplified fragment length of the exogenous insert of the transgenic soybean event SMV-2 is 668bp (FIG. 3 and FIG. 4).

In the invention, the transgenic soybean event SMV-2 specificity detection method adopts a 20 mu L PCR reaction system, and specifically comprises the following steps: 10 XPCR buffer 2. Mu.L, 10mmol/L dNTPs 0.5. Mu.L, 5U/. Mu.L Taq enzyme 0.2. Mu.L, DNA sample 1.0. Mu.L, 10. Mu.mol/L forward primer 0.5. Mu.L, 10. Mu.mol/L reverse primer 0.5. Mu.L, ddH2O 15.3. Mu.L. The PCR reaction conditions were: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s,53 ℃ (left border specific primer)/55 ℃ (right border specific primer) for 30s,72℃for 30s, 32 cycles total; and at 72℃for 5min. The PCR amplification product was checked for the presence of specific bands by 1% agarose gel electrophoresis, and samples were analyzed for the presence of SMV-2 derived components.

The invention has the following technical advantages:

1. the invention discloses flanking sequences of 500bp of the left border and the right border of the exogenous insertion fragment of broad-spectrum anti-mosaic virus transgenic soybean event SMV-2 for the first time, which are respectively described as SEQ ID NO. 1 and SEQ ID NO. 2.

2. The invention analyzes and confirms the left and right boundary flanking sequences of the exogenous insertion fragment of the transgenic soybean event SMV-2 for the first time, comprises the exogenous insertion fragment sequence and a cultivated soybean Tianlong No. one genome sequence (as shown in SEQ ID NO: 5), and determines the specific insertion site of the exogenous fragment in the soybean genome.

3. By using the invention to provide the left boundary and right boundary flanking sequence characteristics of the exogenous insert fragment, a transgenic soybean event SMV-2 specific qualitative PCR detection method is established or a detection kit (specific primer is prepared) is prepared.

4. By utilizing the exogenous insert left and right boundary flanking sequences and the specificity detection method provided by the invention, the specificity detection is carried out on transgenic soybean event SMV-2 including parents, derivative 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.

In summary, the flanking sequences of the exogenous insert fragment of the transgenic soybean event SMV-2 disclosed in the invention can be used as target DNA sequences to establish the transgenic event specific detection method. The exogenous insert fragment flanking sequence and the detection method provided by the invention are suitable for specific detection of transgenic soybean events including parents, derived lines or varieties and products thereof including plants, tissues, seeds and products.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a map of the structure of the SMV-2 transformation vector p7 GWIGG 2-CP.

FIG. 2 is a left border sequence specific PCR assay for T5 generation transgenic soybean SMV-2;

DNAmarker (DL 2000); bk, blank, ddH2O; wt is DNA extracted from wild type, receptor variety Tianlong No. 1, root, stem, leaf, flower and seed of transgenic soybean SMV-2.

FIG. 3 shows the right border sequence-specific PCR assay of T5-generation transgenic soybean SMV-2;

DNAmarker (DL 2000); bk, blank, ddH2O; wt is the receptor variety Tianlong No. 1; t5 transgenic soybean SMV-2 roots, stems, leaves, flowers and seeds.

FIG. 4 PCR detection of the left and right border sequences of transgenic soybean (T5 and T6 generation), transgenic acceptor Tianlong No. 1, tiifeng 31 and maize, rape and wheat.

Wherein the PCR amplification length is 831bp and is the left border amplification sequence length (upper panel), and the PCR amplification length is 668bp and is the right border amplification sequence length (lower panel).

FIG. 5T-schematic representation of the insertion position of DNA at the T5 generation SMV-2.

Detailed Description

In order that the manner in which the above recited features, objects and advantages of the present invention are obtained will become readily apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Example 1 transgenic Soybean event SMV-2 exogenous fragment insertion site analysis

1. Transgenic soybean SMV-2 genomic DNA extraction (conventional technique):

plant genomic DNA was extracted using DNeasy Plant Mini Kit kit (Qiagen, usa) and was performed according to the instructions for use.

(1) Genomic DNA extraction: the DNA of soybean leaves is extracted by adopting a CTAB method. Taking 1-2g of soybean tender leaves, grinding the leaves into powder by liquid nitrogen, and filling the powder into a 50mL centrifuge tube. 5mL of extract A (100 mmol/L Tris-HCl, pH8.0,0.35mol/L sorbitol, 5mmol/L EDTA, pH8.0,1% 2-mercaptoethanol), 3.5mL of extract B (50 mmol/L Tris-HCl, pH8.0,4.0mol/L NaCl,1.8%CTAB,25mmol/LEDTA, pH 8.0), 0.3mL of 30% (volume%) sodium lauroyl sarcosinate and 2% (volume%) PVP-360 were added sequentially, and incubated at 55℃for 60 to 90 minutes with gentle shaking several times. The centrifuge tube was removed, an equal volume of chloroform to isoamyl alcohol (24:1, v/v) was added, the mixture was turned upside down and gently shaken for 15 minutes, and then centrifuged at room temperature for 10 minutes (13000 rpm). The supernatant was aspirated, 2/3 of the pre-chilled volume of isopropanol mixed with 1/10 of the supernatant volume of sodium acetate was added and centrifuged at 13000rpm at 4℃for 20 minutes. The supernatant was discarded and rinsed with cold 75% ethanol. The DNA was dried in air until the surface was dried and stored at-20 ℃.

The preparation method of the extracting solution A comprises the following steps: to 1L of Tris-HCl buffer (10 mmol/L), 0.35mol of sorbitol, 5mmol of EDTA,10ml of 2-mercaptoethanol was added; the pH of the solution was adjusted to 8.0 with either 1mol/L HCL or 1mol/L NaOH.

The preparation method of the extracting solution B comprises the following steps: to 1L of 50mmol/L Tris-HCl buffer, 4.0mol of NaCl, 18g of CTAB (cetyltrimethylammonium bromide), 25mmol of EDTA (ethylenediamine tetraacetic acid) were added, and the pH of the solution was adjusted to 8.0 with 1mol/L HCL or 1mol/L NaOH.

(2) Genomic DNA purification: the DNA was dissolved in 200uL of ddH2O, 5uL of RNase (10 mg/mL) was added, and incubated at 37℃for 40 minutes. The extract was extracted 1-2 times with an 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-chilled 100% chloroform. Centrifuge at 13000rpm for 10 minutes at room temperature. The supernatant was transferred to a new 2mL centrifuge tube, the DNA was precipitated with twice the volume of cold absolute ethanol and 1/10 of 3M sodium acetate solution, and then placed at-20℃for 30 minutes. Centrifugal for 15 minutes at 13000rpm, rinsing with 75% ethanol for 2 times, and drying in air for 15-20 minutes. 50 to 100uLddH2O dissolves DNA. The concentration of DNA was measured by NanoDrop2000 (concentration: about 300-500 ng/. Mu.L), and stored at 20℃for further use.

2. Transgenic soybean SMV-2 genomic resequencing analysis

Re-sequencing transgenic soybean SMV-2 by Shenzhen large gene science and technology service Co., ltd, ultrasonic fragmenting the detected sample genome DNA, purifying the fragmented DNA, and repairing the endAnd A is added at the 3' end, and a sequencing joint is connected. And (3) performing fragment size selection by agarose gel electrophoresis, performing PCR amplification to form a sequencing library, and sequencing the qualified library by adopting a second-generation high-throughput sequencing Xten platform. The T-DNA sequence of the transgenic vector is used as a template, and sequence homology comparison screening (Bowtie 2, http:// Bowtie-bio.sourceforge.net/Bowtie2/index. Shtml, default) is carried out on the whole sequence obtained by sequencing. And further splicing and screening the sequences obtained by screening, and removing the Reads with all the sequences being vector sequences to finally obtain a type of Reads sequences, wherein one half of the Reads are genome sequences and the other half of the Reads are vector sequences. Based on the obtained genome sequence, the genome of the soybean variety williams is obtained on the phytozome websitehttp:// phytozome.jgi.doe.gov/pz/portal.html#！infoalias＝Org_GmaxThe website performs Blast sequence comparison to obtain the specific position of the genome sequence on the genome, namely the possible insertion site. Sequencing gave genome sizes of 979,148,936bp, effective genome sizes of 955,925,112bp (no N in the reference sequence), and a GC content of 33.94% in the reference genome.

Furthermore, for a more smooth analysis, we ligated a number of small fragment Scaffold reference sequences into one or several large chromosomes and renamed them. All samples were aligned between 96% and 96.01% with reference genome of soybean, while the effective sequencing depth was floated between 33.19X and 33.87X. The Clean data is aligned to the insert using BWA, and one aligned upper insert is screened out in combination with the genomic alignment, and the other aligned upper reference genome (pair. Filter screening statistics were performed (filtering is to remove reads with more than 3 mismatches and no complete alignment on ref genome). SMV-2 shares an insert (5513 bp total T-DNA sequence of SEQ ID NO: 5) with the foreign T-DNA insertion site located at the 50509382bp site of chromosome 5 in the forward single copy insertion (FIG. 5).

Example 2 analysis of left and right boundary flanking sequences of exogenous insert for transgenic soybean event SMV-2

PCR detection primers are designed according to the exogenous insertion sequence of transgenic soybean event SMV-2 and the left and right boundary sequences of the insertion site in the soybean reference genome.

The SMV-2 insertion site left border sequence amplification forward primer is 5'-TAATCCATTTTGCCTCGC-3' and reverse primer is 5'-TCAATTCGGCGTTAATTCAG-3', and the expected amplified fragment size is 831bp;

SMV-2 insert right border sequence amplification forward primer 5'-CGTTTCCCGCCTTCAGTT-3' and reverse primer 5'-TGGCGGAATCACCGTAACC-3', with an expected amplified fragment size of 668bp.

PCR amplification was performed using SMV-2 genomic DNA (obtained in step 1 of the example) as a template and the above primers. A20 mu L PCR reaction system is adopted, and the specific steps are as follows: 10 XPCR buffer 2. Mu.L, 10mmol/L dNTPs 0.5. Mu.L, 5U/. Mu.L Taq enzyme 0.2. Mu.L, DNA sample 1.0. Mu.L, 10. Mu.mol/L forward primer 0.5. Mu.L, 10. Mu.mol/L reverse primer 0.5. Mu.L, ddH2O 15.3. Mu.L. The PCR reaction conditions were: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s,53 ℃ (left border specific primer)/55 ℃ (right border specific primer) for 30s,72℃for 30s, 32 cycles total; and at 72℃for 5min. The PCR amplification products were detected by 1% agarose gel electrophoresis. The PCR product was then purified using a gel recovery kit and ligated into the EZ-T cloning vector from GENSTAR company. Sequencing and verification are carried out by entrusting Shanghai, and sequencing results are compared with exogenous insertion sequences and reference genome sequences, so that the transgenic soybean event SMV-2 exogenous insertion fragment left boundary flanking sequence is shown as SEQ ID NO. 1, and right boundary flanking sequence is shown as SEQ ID NO. 2. The sequence consists of a DNA sequence derived from the soybean genomic sequence (SEQ ID NO:1, SEQ ID NO: 2) and from the foreign insert sequence. The left and right border sequences of the foreign insert are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

Example 3 transgenic Soybean event SMV-2 specific PCR detection

Specific detection primers were designed based on the left border flanking sequence (SEQ ID NO: 1) and the right border flanking sequence (SEQ ID NO: 2) of the exogenous insert of the transgenic soybean event SMV-2, respectively.

A500 bp sequence (SEQ ID NO: 1) upstream of the T-DNA insertion site and a 333bp sequence at the left boundary are introduced into software Primer Premier v5.0, a "High" search mode search index is selected, a pair of primers with a rating score higher than 80 and NO "False Primer" is selected, and the predicted amplified fragment is from 498bp upstream to 333bp at the left boundary, which is 831bp in total. The 333bp of the left boundary is shown as SEQ ID NO. 3.

The insert right border Primer was designed in the same way, i.e., a 500bp sequence downstream of the T-DNA insertion site (SEQ ID NO: 2) and a 200bp sequence of the T-DNA right border were introduced into the software Primer Premier v5.0, the "High" search mode search index was selected, a pair of primers with a rating score higher than 80 and NO "False Primer" was selected, and the predicted amplified fragments were 173bp to 495bp downstream of the right border, together 668bp. The 200bp of the right border is shown as SEQ ID NO. 4.

Description: SEQ ID NO. 3 is a sequence of the left border of the foreign insert T-DNA; SEQ ID NO. 1 is to the left of the exogenously inserted T-DNA (a soybean genomic sequence); SEQ ID NO. 4 is a sequence of the right border of the exogenously inserted T-DNA; SEQ ID NO. 2 is to the right of the exogenously inserted T-DNA (a soybean genomic sequence); SEQ ID NO. 5 contains the above sequence.

The invention separately extracts DNA samples of the roots, stems, leaves, flowers and seeds of the transgenic soybean plants SMV-2, and the DNA extraction method is the same as that in example 1. PCR amplification was performed using the acceptor non-transgenic soybean variety Tianlong No. 1, soybean variety Tiifeng 31 and maize, canola and wheat as controls.

For left border augmentation:

the primer is 5'-TAATCCATTTTGCCTCGC-3' and the reverse primer is 5'-TCAATTCGGCGTTAATTCAG-3', and the expected amplified fragment size is 831bp;

the amplification system was a 20. Mu.L PCR reaction system containing 2. Mu.L of 10 XPCR buffer, 0.5. Mu.L of 10mmol/L dNTPs, 0.2. Mu.L of 5U/. Mu.L Taq enzyme, 1.0. Mu.L of DNA sample, 0.5. Mu.L of 10. Mu.mol/L forward primer, 0.5. Mu.L of 10. Mu.mol/L reverse primer, and 15.3. Mu.L of ddH 2O. The amplification procedure was 94℃for 2min of pre-denaturation; denaturation at 94℃for 30s, denaturation at 53℃for 30s and denaturation at 72℃for 30s for 32 cycles; and at 72℃for 5min.

For right border augmentation:

the primer is 5'-CGTTTCCCGCCTTCAGTT-3' and the reverse primer is 5'-TGGCGGAATCACCGTAACC-3', and the expected amplified fragment size is 668bp;

the amplification system is a 20 mu L PCR reaction system, and comprises 2 mu L of 10 XPCR buffer solution, 0.5 mu L of 10mmol/L dNTPs, 0.2 mu L of 5U/mu L Taq enzyme, 1.0 mu L of DNA sample, 0.5 mu L of 10 mu mol/L forward primer, 0.5 mu L of 10 mu mol/L reverse primer and 15.3 mu L of ddH2O, and the amplification program is pre-denatured for 2min at 94 ℃; denaturation at 94℃for 30s, denaturation at 55℃for 30s and denaturation at 72℃for 30s for 32 cycles; and at 72℃for 5min.

The PCR products were separated by 1% agarose gel electrophoresis and stained with EB to identify the presence of specific amplified bands. When the specific primers are used for PCR amplification, the non-transgenic soybean variety Tianlong No. I, fengfeng 31, corn, rape and wheat have no amplified bands, and only transgenic soybean SMV-2 samples comprise roots, stems, leaves, flowers and seeds to generate specific amplified bands. Wherein the length of the amplified fragment of the left boundary flanking sequence is 831bp, as shown in fig. 2 and 4; the amplified fragment length of the right border flanking sequence is 668bp, as shown in FIGS. 3 and 4. This study shows that PCR analysis using primers specific for flanking sequences of the exogenous insert can specifically detect whether the sample contains SMV-2 derived components.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Sequence listing

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tgcggctcgg tacggaagtt gaccgtgctt gtctcgatgt agtggttgac gatggtgcag 1620

accgccggca tgtccgcctc ggtggcacgg cggatgtcgg ccgggcgtcg ttctgggctc 1680

atggtagatc ccctcgatcg agttgagagt gaatatgaga ctctaattgg ataccgaggg 1740

gaatttatgg aacgtcagtg gagcattttt gacaagaaat atttgctagc tgatagtgac 1800

cttaggcgac ttttgaacgc gcaataatgg tttctgacgt atgtgcttag ctcattaaac 1860

tccagaaacc cgcggctcag tggctccttc aacgttgcgg ttctgtcagt tccaaacgta 1920

aaacggcttg tcccgcgtca tcggcggggg tcataacgtg actcccttaa ttctcatgta 1980

tgataattcg agggtacccg gggatcctct agagggcccg acgtcgcatg cctgcaggtc 2040

actggatttt ggttttagga attagaaatt ttattgatag aagtatttta caaatacaaa 2100

tacatactaa gggtttctta tatgctcaac acatgagcga aaccctataa gaaccctaat 2160

tcccttatct gggaactact cacacattat tctggagaaa aatagagaga gatagatttg 2220

tagagagaga ctggtgattt ttgcggactc tagcatggcc gcgggatatc acaagtttgt 2280

acaaaaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt 2340

tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca ctatggttcg 2400

ccgaacttgg tctcaactac aactttgagg aacggacaaa gaaaagggag gaattgtggt 2460

tcatgtcaca ccaagccatg ttagttgatg gaatctatat tccaaaactt gaacctgaga 2520

gaattgtctc tatcctagag tgggacagga gcaaagagct tatgcatcgc actgaggcga 2580

tatgcgcagc aatgattgag gcatggggat acactgaatt gctgcaagag atccgcaaat 2640

tttatttgtg gcttctaaac aaggatgaat ttaaagagct tgcttcgtct ggaaaagcac 2700

catatattgc agagacagct ttgagaaagc tgtacacaga tgtcaatgct cagacaagtg 2760

agctacaaag atatcttgaa gtgctggatt tcactcatgc tgatgactgt tgtgaatcag 2820

tgtccttaca atcaggcaag gagaaggaag gagaaatgga tgcaggtaag gatccaaaga 2880

agagcaccag cagtagcaaa ggagctggta caagcagcaa agatgtaaat gttggatcaa 2940

aaggaaaggt ggttccgcgt ttgcagaaga ttacaagaaa gatgaatctt ccaatggtcg 3000

aagggaagat cacatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt 3060

ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct 3120

ttcttgtaca aagtggtgat atcactagtg cggccgcctg caggtcgacc atatggtcga 3180

cctgcaggcg gccgcactag tgatgctgtt atgttcagtg tcaagctgac ctgcaaacac 3240

gttaaatgct aagaagttag aatatatgag acacgttaac tggtatatga ataagctgta 3300

aataaccgag tataaactca ttaactaata tcacctctag agtataatat aatcaaattc 3360

gacaatttga ctttcaagag taggctaatg taaaatcttt atatatttct acaatgttca 3420

aagaaacagt tgcatctaaa cccctatggc catcaaattc aatgaacgct aagcttaata 3480

tgactctcaa taaagtctca taccaacaag tgccacctta ttcaaccatc aagaaaaaag 3540

ccaaaattta tgctactcta aggaaaactt cactaaagaa gacgatttag agtgttttac 3600

caagaatttc tgtcatctta ctaaacaact aaagatcggt gtgatacaaa acctaatctc 3660

attaaagttt atgctaaaat aagcataatt ttacccacta agcgtgacca gataaacata 3720

actcagcaca ccagagcata tatattggtg gctcaaatca tagaaactta cagtgaagac 3780

acagaaagcc gtaagaagag gcaagagtat gaaaccttac ctcatcattt ccatgaggtt 3840

gcttctgatc ccgcgggata tcaccacttt gtacaagaaa gctgaacgag aaacgtaaaa 3900

tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg 3960

taaaacacaa catatccagt cactatgact agaagggaag ctggtaacct tctaagtaga 4020

aagaacatta gaagacgttt gcgccttggt ggaaaggaaa actaggttgt aaatgtagaa 4080

acgacgaaca tggtcgagga aacgatgacg accacgagaa gaaacctagg aatggacgta 4140

ggtaaagagg aaggaagagg aacggactaa cattcctgtg actaagtgtt gtcagtagtc 4200

gtactcactt taggtcgtga agttctatag aaacatcgag tgaacagact cgtaactgta 4260

gacacatgtc gaaagagttt cgacagagac gttatatacc acgaaaaggt ctgcttcgtt 4320

cgagaaattt aagtaggaac aaatcttcgg tgtttatttt aaacgcctag agaacgtcgt 4380

taagtcacat aggggtacgg agttagtaac gacgcgtata gcggagtcac gctacgtatt 4440

cgagaaacga ggacagggtg agatcctatc tctgttaaga gagtccaagt tcaaaacctt 4500

atatctaagg tagttgattg taccgaacca cactgtactt ggtgttaagg agggaaaaga 4560

aacaggcaag gagtttcaac atcaactctg gttcaagccg cttgcatagt gactggatat 4620

gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga 4680

tatttatatc attttacgtt tctcgttcag cttttttgta caaacttgtg atatcactag 4740

tgcggccgcc tgcaggtcga ctagaatagt aaattgtaat gttgtttgtt gtttgttttg 4800

ttgtggtaat tgttgtaaaa atacggatcg tcctgcagtc ctctccaaat gaaatgaact 4860

tccttatata gaggaagggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 4920

agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 4980

cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 5040

aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 5100

tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 5160

taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 5220

cttggagtag acgagagtgt cgtgctccac catgttgacg aagattttct tcttgtcatt 5280

gagtcgtaaa agactctgta tgaactgttc gccagtcttc acggcgagtt ctgttagatc 5340

ctcgatctga atttttgact ccatggcctt tgattcagta ggaactactt tcttagagac 5400

tccaatctct attacttgcc ttggtttatg aagcaagcct tgaatcgtcc atactggaat 5460

agtacttctg atcttgagaa atatatcttt ctctgtgttc ttgatgcagt tagtcctgaa 5520

tcttttgact gcatctttaa ccttcttggg aaggtatttg atctcctgga gattattact 5580

cgggtagatc gtcttgatga gacctgccgc gtaggcctct ctaaccatct gtgggtcagc 5640

attctttctg aaattgaaga ggctaatctt ctcattatcg gtggtgaaca tggtatcgtc 5700

accttctccg tcgaactttc ttcctagatc gtagagatag agaaagtcgt ccatggtgat 5760

ctccggggca aaggagatca gcttggctct agtcgaccat atgggagagc tcaagcttag 5820

cttgagcttg gatcagattg tcgtttcccg ccttcagttt aaactatcag tgtttgacag 5880

gatatattgg cgggtaaacc taagagaaaa gagcgtttat tagaataacg gatatttaaa 5940

agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc acagggttcc 6000

cctcgggatc aaaactggat ctttcccggg tgtacagcaa tcgacggtag agatgagccc 6060

ctatggtgta tccttcgtgg gtaccgtgga ccatacccca ccatgtatga catcttcaat 6120

gggttgtctc caagcaagta atccacctga aaatacattt ttatttttat tttaaaaaaa 6180

aaatcaagtt caattaacaa gaataggaaa tcaagcaaca cattttaata gattaaaata 6240

tattgaaccc atttttttat gggacaaata ataattatta gtttgtaatt ttttattaac 6300

ggaaaaagtt ggaatccgtc acccttttct tattattatt attattatta tttttttaat 6360

catctaatca atatctccca aatatattga acatataata catgattcga ttattactgc 6420

ataaaaagtt tatttttttt cttcttcttt ctcgaacctg tttcttagct attgtccgga 6480

gtctccttgg ggttacggtg attccgccac agt 6513

Claims (6)

1. Use of an anti-mosaic virus transgenic soybean event SMV-2 exogenous insert flanking a test sample for detecting whether the test sample contains a component derived from SMV-2, characterized in that:

500bp flanking the left border is shown in SEQ ID NO. 1; 500bp flanking the right border is shown in SEQ ID NO. 2.

2. Use according to claim 1, characterized in that: the DNA sequence consisting of the sequence derived from soybean genome and the sequence derived from exogenous insert is shown in SEQ ID NO. 5.

3. The use according to claim 2, characterized in that said anti-mosaic virus transgenic soybean event SMV-2 exogenous insert flanking is prepared by: the transgenic soybean event SMV-2 genomic DNA is extracted, the exogenous fragment insertion site is determined by utilizing the analysis of a genome re-sequencing technology, and the exogenous fragment insertion site is obtained by PCR amplification.

4. Use according to claim 3, characterized in that:

the SMV-2 left border detection primers were:

the forward primer is as follows: LB-F1, 5'-TAATCCATTTTGCCTCGC-3', the total number of the components,

the reverse primer is as follows: LB-R1:5'-TCAATTCGGCGTTAATTCAG-3';

when 831bp fragments are obtained through amplification, judging that the sample to be detected contains components derived from SMV-2; otherwise, it is not contained.

5. Use according to claim 4, characterized in that:

the SMV-2 right border detection primer is:

the forward primer is as follows: 5'-CGTTTCCCGCCTTCAGTT-3' the number of the individual pieces of the plastic,

the reverse primer is as follows: 5'-TGGCGGAATCACCGTAACC-3';

when 668bp fragment is obtained by amplification, determining that the sample to be detected contains a component derived from SMV-2; otherwise, it is not contained.

6. Use according to claim 4 or 5, characterized in that:

the sample to be tested comprises plants, tissues and seeds corresponding to the parent strain, the derivative strain or the variety of the SMV-2.