CN112359038A - Cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanking sequence and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanking sequence and application thereof. The invention discloses a cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flank, 500bp of the left boundary flank is as shown in SEQ ID NO. 1; the 500bp flanking the right border is as shown in SEQ ID NO. 2. Extracting genome DNA of the transgenic soybean event ZHs1-2, analyzing and determining an insertion site of an exogenous fragment by using a genome re-sequencing technology, and obtaining the transgenic soybean event by PCR amplification. The purpose is to detect whether the sample to be detected contains components derived from ZHs 1-2.

Description

Cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanking sequence and application thereof

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to a cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanking sequence and application thereof.

Background

The detection of the specific transformation event of the transgenic plant depends on the analysis of the flanking sequence of the insertion site of the foreign gene and designs corresponding primers according to the known flanking sequence. Therefore, it is important to clarify the information of the insertion site of T-DNA for the smooth development of the related studies. In the application of transgene production, the insertion site of the exogenous gene is a label of each gene transformation event, and the screening and identification of the transformation event, the safety evaluation of the transformation event, the environmental release application and the like all require the T-DNA insertion site of each gene transformation event and the flanking sequence of the inserted gene.

There are many methods for obtaining flanking sequences of a T-DNA insertion site, and the methods can be classified into 3 types according to the principle: reverse 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 the identification of insertion sites and the acquisition of flanking sequences. However, these methods have disadvantages of complicated operation steps, long time consumption, etc., and also have great uncertainty, poor specificity, and no high success rate.

In recent years, with the development of sequencing technology, the whole genome re-sequencing technology is becoming mature. The time and cost of whole genome re-sequencing are greatly reduced, so that the application of whole genome re-sequencing is more and more extensive, and the method is not only used for determining whole genome sequences of different species and constructing whole genome maps, but also used for the aspects of mutant gene cloning, insertion site flanking sequence analysis and the like.

Transgenic soybean event ZHs1-2 is the introduction of exogenous Hs1 by Agrobacterium mediated method^pro-1The gene is transferred into a cultivated soybean variety Tianlong I. ZHs1-2 the transformation vector is pHs1, and the structure map is shown in FIG. 1. The carrier contains Hs1^pro-1An open reading frame between 277 bp-1122 bp of gene, 846bp, coding phosphinothricin acetyl transferase gene, BAR gene, as screening marker gene, showing the resistance to glufosinate herbicide, and the used agrobacterium strainIs EHA 105. The transformation vector pHs1 and the transgenic soybean event ZHs1-2 are both completed by the university of Zhejiang crop science institute in 2013, and details about vector construction and the acquisition of the transformation event are found in a Liangyan written Master paper, germplasm innovation research on cyst nematode resistant transgenic soybeans (2013).

Disclosure of Invention

The invention aims to provide a flanking sequence of an exogenous insert of cyst nematode resistant transgenic soybean event ZHs1-2 and application thereof.

In order to solve the technical problem, the invention provides a cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flank, 500bp of the left boundary flank is as shown in SEQ ID NO. 1; the 500bp flanking the right border is as shown in SEQ ID NO. 2.

As an improvement flanking the exogenous insert of cyst nematode resistant transgenic soybean event ZHs1-2 of the present invention: DNA sequence composed of soybean genome sequence and exogenous insert sequence, as shown in SEQ ID NO. 5;

the 1 st-500 th site sequence of SEQ ID NO. 5 is derived from a cultivated soybean Tianlong No. 1 genome sequence (SEQ ID NO. 1), the 501 st and 5476 th site sequences are derived from an exogenous T-DNA insert sequence, and the 5477 st and 5976 th site sequences are derived from a cultivated soybean Tianlong No. 2 genome sequence;

therefore, SEQ ID NO. 5 is an exogenous insert and the left and right flanking sequences of the insert, which are 5976bp in total; 1 is a soybean genome flanking sequence of the exogenous T-DNA inserted into the left side of the soybean genome, and the 501-5476 site sequence is a T-DNA sequence of the exogenous T-DNA inserted into the soybean genome; SEQ ID NO 2 is the soybean flanking sequence to the right of the T-DNA insertion site, i.e., the 5477-5976 site sequence.

The invention also provides a preparation method of the flanks of the exogenous insert of the cyst nematode-resistant transgenic soybean event ZHs1-2, which comprises the following steps: extracting genome DNA of the transgenic soybean event ZHs1-2, analyzing and determining an insertion site of an exogenous fragment by using a genome re-sequencing technology, and obtaining the transgenic soybean event by PCR amplification.

The invention also provides the application of the exogenous insert flank of the cyst nematode resistant transgenic soybean event ZHs 1-2: and detecting whether the sample to be detected contains the component derived from ZHs 1-2.

As an improvement of the use of the present invention:

ZHs1-2 the left border detection primers were:

the forward primer LB-F1 is: f, 5'-ATGCACTATTCAAATAGGAGCA-3', and the method comprises the following steps of,

the reverse primer LB-R1 is: 5'-ATTCAGTACATTAAAAACGTCCG-3', respectively;

the expected amplified fragment size was 821 bp.

That is, when the 821bp fragment was obtained by amplification, it was judged that the component derived from ZHs1-2 was contained in the sample. Otherwise, it does not.

As an improvement of the use of the present invention:

ZHs1-2 the right border detection primers were:

the forward primer is: 5'-CCCGATCGTTCAAACATT-3' the flow of the air in the air conditioner,

the reverse primer is: 5'-CTCATCTCCTCTTATGAATAGACTA-3', respectively;

the expected amplified fragment size is 1184 bp.

That is, when this 1184bp fragment was obtained by amplification, it was judged that the component derived from ZHs1-2 was contained in the sample to be tested. Otherwise, it does not.

The ZHs1-2 left border detection primer or ZHs1-2 right border detection primer can be selected and judged according to the result. The two are carried out simultaneously, so that the detection result can be ensured to be more accurate.

As an improvement of the use of the present invention:

the sample to be detected comprises plants, tissues, seeds and products corresponding to the parents, derived strains or varieties of ZHs 1-2.

In the present invention:

according to the specificity detection primers of the left border flanking sequence of the ZHs1-2 exogenous insert (ZHs1-2 left border detection primers), one primer is a forward primer designed according to the sequence of the 1 st-500 th site of SEQ ID NO. 5, and the other primer is a reverse primer designed according to the sequence of the 501 th-821 th site of SEQ ID NO. 5.

According to the specific detection primers (ZHs1-2 right border detection primers) of the flanking sequences at the right border of the ZHs1-2 exogenous insert, one primer is a forward primer designed according to the sequence of the 4769-5476 site of SEQ ID NO. 5, and the other primer is a reverse primer designed according to the sequence of the 5477-5976 site of SEQ ID NO. 5.

In the invention, DNA samples of roots, stems, leaves, flowers and seeds of a T3 generation transgenic soybean event ZHs1-2 are respectively extracted, and PCR amplification is carried out by taking a receptor non-transgenic soybean variety Tianlong I as a control. 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 ZHs1-2 exogenous insertion fragment is 821bp (FIG. 2 and FIG. 3), and the length of the right border amplification fragment of the transgenic soybean event ZHs1-2 exogenous insertion fragment is 1184bp (FIG. 2 and FIG. 3).

In the invention, the specific detection method of the transgenic soybean event ZHs1-2 adopts a 20 mu L PCR reaction system, which specifically comprises the following steps: 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 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 ddH2O 15.3. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, 54 ℃ (left border specific primer)/52 ℃ (right border specific primer) for 30s, 72 ℃ for 30s, 32 cycles; 5min at 72 ℃. The presence of a specific band in the PCR amplification product was detected by 1% agarose gel electrophoresis, and the sample was analyzed for the presence of ZHs 1-2-derived components.

The invention has the following technical advantages:

1. the invention discloses flanking sequences of 500bp of the left boundary and the right boundary of a broad-spectrum anti-mosaic virus transgenic soybean event ZHs1-2 exogenous insertion fragment for the first time, which are respectively shown 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 ZHs1-2 for the first time, comprises an exogenous insertion fragment sequence and a cultivated soybean Tianlong No. I genome sequence (as shown in SEQ ID NO: 5), and determines the specific insertion site of the exogenous fragment in the soybean genome.

3. The invention provides the characteristics of the flanking sequences of the left boundary and the right boundary of the exogenous insertion fragment, and establishes a specific qualitative PCR detection method (comprising the preparation of specific primers) for the transgenic soybean event ZHs 1-2.

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 ZHs1-2 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.

In summary, the flanking sequences of the exogenous insert of transgenic soybean event ZHs1-2 disclosed by the invention can be used as target DNA sequences 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.

Drawings

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

FIG. 1 is a diagram of Hs1 expression vector pHs1 of transgenic soybean ZHs1-2 obtained by Agrobacterium mediation.

The expression vector contains a T-DNA region and an agrobacterium plasmid skeleton region, wherein the T-DNA region contains a target gene Hs1 expression frame and a Bar gene expression frame, and the Bar gene expression frame can encode phosphinothricin acetyltransferase and is used as a transformation screening marker to express the resistance to herbicide glufosinate; the backbone region of the agrobacterium plasmid contains aadA gene and encodes aminoglycoside adenylyltransferase, which confers spectinomycin resistance to agrobacterium. The agrobacterium strain used was EHA 105.

FIG. 2 shows specific PCR detection of the left border sequence (upper panel, 821bp) and the right border sequence (lower panel, 1184bp) of T3 and T4 transgenic soybean ZHs 1-2.

M is DNA marker (DL 2000); bk blank control, ddH₂O; wt, wild type, receptor variety Tianlong No. one; transformation of root, stem, leaf, flower and seed extracted DNA of event ZHs 1-2.

FIG. 3 shows the PCR detection of the gene ZHs1-2 transgenic soybean (T3 and T4 generation), transgenic recipient variety Tianlong I, Tiefeng 31 and the left and right border sequence specificity of corn, rape and wheat.

Wherein, the PCR amplification length of 821bp is the length of the left margin amplification sequence (upper graph), and the PCR amplification length of 1184bp is the length of the right margin amplification sequence (lower graph).

FIG. 4 is a schematic diagram showing the insertion position of T-DNA in soybean genome. The T-DNA is inserted into the soybean genome in a single copy positive orientation at the 16184657 th site on chromosome 6.

Detailed Description

The present invention will be further described with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easily understood, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. 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 ZHs1-2 exogenous fragment insertion site analysis

1. Transgenic soybean ZHs1-2 genomic DNA extraction (conventional technique):

(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% (vol%) sodium lauroyl sarcosinate and 2% (vol%) PVP-360 were added in this order, incubated at 55 ℃ for 60 to 90 minutes, while shaking gently. The tube was removed, added with an equal volume of chloroform/isoamyl alcohol (24:1, v/v), 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. The DNA was air-dried to surface-dry and stored at-20 ℃.

The preparation method of the extracting solution A comprises the following steps: adding 0.35mol of sorbitol, 5mmol of EDTA and 10ml of 2-mercaptoethanol into 1L of 10mmol/L Tris-HCl buffer solution; the pH of the solution was adjusted to 8.0 with 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 solution were added 4.0mol of NaCl, 18g of CTAB (cetyltrimethylammonium bromide), 25mmol of EDTA (ethylenediaminetetraacetic acid), and the pH of the solution was adjusted to 8.0 with 1mol/L HCl or 1mol/L NaOH.

(2) And (3) genomic DNA purification:

with 200. mu.L 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, two volumes of cold absolute ethanol and 1/10 volumes of 3M sodium acetate solution were added, 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 uLddH₂O dissolves the DNA. After the DNA concentration is measured by the NanoDrop2000 (the concentration is about 300-500 ng/uL), the DNA is stored at the temperature of-20 ℃ for later use.

2. Transgenic soybean ZHs1-2 genome re-sequencing analysis

The Shenzhen Hua Dagen science and technology service Limited company is entrusted with performing resequencing analysis on the transgenic soybean ZHs1-2, fragmenting qualified sample genome DNA by using ultrasonic waves, and then purifying, repairing the tail end, adding A at the 3' end and connecting a sequencing joint to the fragmented DNA. And then, agarose gel electrophoresis is used for fragment size selection, PCR amplification is carried out to form a sequencing library, and the library qualified by quality inspection is sequenced by adopting a second-generation high-throughput sequencing Xten platform. The T-DNA sequence of the transgenic vector is used as a template, and the sequence homology comparison and screening is carried out with the whole sequence obtained by sequencing (Bowtie2, http:// Bowtie-Net/bowtie2/index shtml, default setting). And further splicing and screening the screened sequences, and removing Reads with all sequences as carrier sequences to finally obtain a class of Reads sequences, which is characterized in that half of the class of Reads sequences is a genome sequence, and the other half of the class of Reads sequences is a carrier sequence. According to the obtained genome sequence, the genome of the soybean variety williams is located in the phytozome websitehttp://phytozome.jgi.doe.gov/pz/portal.html#！infoalias＝Org_ GmaxAnd performing Blast sequence comparison on the website to obtain a specific position of the genome sequence on the genome, namely a possible insertion site. The genome size obtained by sequencing is 979,148,936bp, the effective genome size is 955,925,112bp (the reference sequence does not contain N), and the GC content of the reference genome is 33.94%.

In addition, for a more smooth analysis, a large number of small fragment Scaffold reference sequences were ligated into one or several large chromosomes and renamed. The alignment rates (to soybean reference genome) for all samples were between 96% and 96.01%, while the effective sequencing depth floated between 33.19X and 33.87X. And comparing the Clean data to the insert by using BWA, and screening out an insert in comparison and a reference genome in comparison (a. pair) by combining the genome comparison result. Filter screening statistics were performed (filters were done to remove more than 3 mismatches and not perfectly aligned reads on the ref genome). ZHs1-2 share an insert (4976 bp, T-DNA sequence in SEQ ID NO: 5) which is inserted into the transformation event, the insertion position renamed ZHs1-2-insert, ZHs1-2-insert is located at the 16184657bp site on chromosome 6 approximately at the foreign T-DNA insertion position, in the forward single copy insertion orientation (FIG. 4).

The ZHs1-2-insert is shown as SEQ ID NO:5, and the total length is 5976 bp; i.e.the exogenously inserted T-DNA sequence and the left and right flanking sequences of the T-DNA are each 500 bp.

Example 2 analysis of the left and right border flanking sequences of the exogenous insert of transgenic Soybean event ZHs1-2

And designing PCR detection primers according to the left flanking sequence and the right flanking sequence of the exogenous insertion sequence and the insertion site of the transgenic soybean event ZHs1-2 in the soybean reference genome.

ZHs1-2 insertion site left border sequence amplification forward primers were F: 5'-ATGCACTATTCAAATAGGAGCA-3' and 5'-ATTCAGTACATTAAAAACGTCCG-3' (Tm: 54 ℃), with an expected amplified fragment size of 821 bp;

ZHs1-2 insertion right border sequence the amplification forward primer was 5'-CCCGATCGTTCAAACATT-3' and the reverse primer was 5'-CTCATCTCCTCTTATGAATAGACTA-3' (Tm 52 ℃), with an expected amplified fragment size of 1184 bp.

The genomic DNA (obtained in step 1 of the example) of ZHs1-2 was used as a template, and PCR amplification was carried out using the above primers. A20-mu-L PCR reaction system is adopted, and specifically comprises the following steps: 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 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 ddH2O 15.3. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, 54 ℃ (left border specific primer)/52 ℃ (right border specific primer) for 30s, 72 ℃ for 30s, 32 cycles; 5min at 72 ℃. 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 left flank sequence of the exogenous insertion fragment of the transgenic soybean event ZHs1-2, wherein the left flank sequence is shown as SEQ ID NO. 1, and the right flank sequence is shown as SEQ ID NO. 2. The sequence is a DNA sequence consisting of a soybean genome sequence (SEQ ID NO:1 and SEQ ID NO:2) and an exogenous insert sequence. The sequences of the left border and the right border of the exogenous insert are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

Example 3 transgenic Soybean event ZHs1-2 specific PCR detection

Specific detection primers were designed based on the left (SEQ ID NO:1) and right (SEQ ID NO:2) flanking sequences of the exogenous insert of transgenic soybean event ZHs1-2, respectively. The sequence 500bp upstream of the T-DNA insertion site (SEQ ID NO:1) and the sequence 321bp left border were introduced into the software Primer Premier v5.0, a "High" search mode search Primer was selected, a pair of primers with a rating score higher than 80 and NO "False prime" was selected, and the amplified fragment was predicted to be 500bp upstream to 321bp left border, 821bp in total. The left border 321bp is shown in SEQ ID NO 3.

Description of the drawings: SEQ ID NO. 3 is the left border sequence of T-DNA in which exogenous T-DNA is inserted into transgenic soybean ZHs1-2, SEQ ID NO. 1 is the flanking sequence located on the left side of T-DNA, the flanking sequence is a sequence derived from soybean genome, 1-500bp in SEQ ID NO. 5 is the sequence of SEQ ID NO. 1, and 501bp-821bp in SEQ ID NO. 5 is the sequence of SEQ ID NO. 3.

Designing a right boundary Primer of the inserted segment according to the same method, namely, introducing a downstream 500bp sequence and a right boundary 708bp sequence of the T-DNA insertion site into software Primer Premier v5.0, selecting a High search mode search Primer, selecting a pair of primers with the rating score higher than 80 and without False prime, and predicting that the amplified segment is from the right boundary 708bp to the downstream 476bp, and the total length is 1184 bp. The right border 708bp is shown in SEQ ID NO. 4.

Description of the drawings: SEQ ID NO. 4 is the right border sequence of T-DNA where exogenous T-DNA is inserted into transgenic soybean ZHs1-2, SEQ ID NO. 2 is the right flank sequence where the T-DNA is inserted, the flank sequence is derived from soybean genome, 4769-5476bp in SEQ ID NO. 5 is the sequence of SEQ ID NO. 4, and 5477-5976 bp in SEQ ID NO. 5 is the sequence of SEQ ID NO. 2.

DNA samples of roots, stems, leaves, flowers and seeds of transgenic soybean plant ZHs1-2 were extracted, respectively, using the DNA extraction method as described in example 1. PCR amplification was performed with the recipient non-transgenic soybean variety Tianlong No. 1, soybean variety Tiefeng 31 and corn, canola and wheat as controls.

When amplified against the left border:

the primers are a forward primer 5'-ATGCACTATTCAAATAGGAGCA-3' and a reverse primer 5'-ATTCAGTACATTAAAAACGTCCG-3' (Tm: 54 ℃), and the expected amplified fragment size is 821 bp;

the amplification system is a 20 mu L PCR reaction system, which specifically comprises the following steps: 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 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 ddH2O 15.3. The amplification procedure was: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, at 54 ℃ for 30s, and at 72 ℃ for 30s for 32 cycles; 5min at 72 ℃.

When amplified against the right border:

the primers were forward 5'-CCCGATCGTTCAAACATT-3' and reverse 5'-CTCATCTCCTCTTATGAATAGACTA-3' (Tm 52 ℃), and the expected amplified fragment size was 1184 bp.

The amplification system is a 20 mu L PCR reaction system, which specifically comprises the following steps: 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 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 ddH2O 15.3. The amplification procedure was 94 ℃ pre-denaturation for 2 min; denaturation at 94 ℃ for 30s, denaturation at 52 ℃ (30s, and denaturation at 72 ℃ for 30s, 32 cycles, and denaturation at 72 ℃ for 5 min.

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, no amplification band exists in the non-transgenic soybean varieties of Tianlong I, Tiefeng 31, corn, rape and wheat, and only the transgenic soybean ZHs1-2 sample comprising roots, stems, leaves, flowers and seeds generates a specific amplification band. Wherein the length of the amplified fragment of the left border flanking sequence is 821bp, as shown in FIG. 2 and FIG. 4; the amplified fragment of the right border flanking sequence was 1184bp in length, as shown in FIGS. 3 and 4. The present study shows that PCR analysis using primers specific for the flanking sequences of the foreign insert can specifically detect whether the sample contains components derived from ZHs 1-2.

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.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

<110> Zhejiang university

<120> cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanking sequence and uses thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgcactatt caaataggag cattagctat gtttgttaat gtcactttat gttatgtggg 60

taagtcacct aagacactcc acgtacctac ttgttgtctc ttacgcggct ttaataaatc 120

ttctgccctt gttccatatt tactaattat ccctttcttc actaaaagaa aattgttatc 180

attaagtatt agtctttaga acatatgagg tctttaattg ggtaggtttt acaaattaac 240

taatataaaa tgtcataaaa tccacgtggt taaacaaatg cagaaaatcg acgtcgtcta 300

ttggaccgac agttgctatt aatataatgg gccaccatag tagactgaca aataaattac 360

ctgacaacat cgtttcacaa aaaaacaaac acaaaaaggg agtgcatttt ccagggcatt 420

tttgtaataa aaaacagtta aaagggagtg caatagaaat ataggggtgt ggaaatagtg 480

atttgagcac gtcttgaagc 500

<210> 2

<211> 500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aaattgggtt gttcagtttt gggctggccg aagaaaatta aaagatagtg gagctcaacc 60

caactaattt tcttctattc cactttaatc taaccctcaa ttaattagtt taattttatg 120

actaattttt ttaatgcaca tacaatgaat tctaaaaatt tacttcacac acaaactatc 180

aatttatata aattcattat acttatttca aaagcgaact aattataaac tctttgcatt 240

atttcaaaag cgaactaatt ataaactctt tgcattattt tggtttagct agaaatacat 300

gcaattccac aaatagttac aaacacagtt attcgagtga tggatggatg gatgtctcaa 360

ttatggctgc acaaatacac gagttgtgcc ttagggagag ggggcggggg aatcatgctt 420

ttgggtggaa aaaattcatt tctaatgctt tgtctgtttc acctgaaaag ataattagtc 480

tattcataag aggagatgag 500

<210> 3

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga 60

tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat 120

gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct 180

gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg 240

tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 300

gacgttttta atgtactgaa t 321

<210> 4

<211> 708

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctagannngg atcccccgaa tttccccgat cgttcaaaca tttggcaata aagtttgttt 60

cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 120

cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 180

gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 240

ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tcgtaatcat 300

gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 360

cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 420

gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 480

cggccaacgc gcggggagag gcggtttgcg tattggagct tgagcttgga tcagattgtc 540

gtttcccgcc ttcagtttaa actatcagtg tttgacagga tatattggcg ggtaaaccta 600

agagaaaaga gcgtttatta gaataatcgg atatttaaaa gggcgtgaaa aggtttatcc 660

gttcgtccat ttgtatgtgc atgccaacca cagggttccc ctcgggat 708

<210> 5

<211> 5976

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgcactatt caaataggag cattagctat gtttgttaat gtcactttat gttatgtggg 60

taagtcacct aagacactcc acgtacctac ttgttgtctc ttacgcggct ttaataaatc 120

ttctgccctt gttccatatt tactaattat ccctttcttc actaaaagaa aattgttatc 180

attaagtatt agtctttaga acatatgagg tctttaattg ggtaggtttt acaaattaac 240

taatataaaa tgtcataaaa tccacgtggt taaacaaatg cagaaaatcg acgtcgtcta 300

ttggaccgac agttgctatt aatataatgg gccaccatag tagactgaca aataaattac 360

ctgacaacat cgtttcacaa aaaaacaaac acaaaaaggg agtgcatttt ccagggcatt 420

tttgtaataa aaaacagtta aaagggagtg caatagaaat ataggggtgt ggaaatagtg 480

atttgagcac gtcttgaagc attgatcaca ggcagcaacg ctctgtcatc gttacaatca 540

acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt 600

ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg 660

ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg 720

gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt gacgcttaga 780

caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga attgctctag 840

cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 900

ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 960

ttttcccagt cacgacgttg taaaacgacg gccagtgcca agctaattcg cttcaagacg 1020

tgctcaaatc actatttcca cacccctata tttctattgc actccctttt aactgttttt 1080

tattacaaaa atgccctgga aaatgcactc cctttttgtg tttgtttttt tgtgaaacga 1140

tgttgtcagg taatttattt gtcagtctac tatggtggcc cattatatta atagcaactg 1200

tcggtccaat agacgacgtc gattttctgc atttgtttaa ccacgtggat tttatgacat 1260

tttatattag ttaatttgta aaacctaccc aattaaagac ctcatatgtt ctaaagacta 1320

atacttaatg ataacaattt tcttttagtg aagaaaggga taattagtaa atatggaaca 1380

agggcagaag atttattaaa gccgcggtaa gagacaacaa gtaggtacgt ggagtgtctt 1440

aggtgactta cccacataac ataaagtgac attaacaaac atagctaatg ctcctatttg 1500

aatagtgcat atcagcatac cttattacat atagatagga gcaaactcta gctagattgt 1560

tgagcagatc tcggtgacgg gcaggaccgg acggggcggt accggcaggc tgaagtccag 1620

ctgccagaaa cccacgtcat gccagttccc gtgcttgaag ccggccgccc gcagcatgcc 1680

gcggggggca tatccgagcg cctcgtgcat gcgcacgctc gggtcgttgg gcagcccgat 1740

gacagcgacc acgctcttga agccctgtgc ctccagggac ttcagcaggt gggtgtagag 1800

cgtggagccc agtcccgtcc gctggtggcg gggggagacg tacacggtcg actcggccgt 1860

ccagtcgtag gcgttgcgtg ccttccaggg gcccgcgtag gcgatgccgg cgacctcgcc 1920

gtccacctcg gcgacgagcc agggatagcg ctcccgcaga cggacgaggt cgtccgtcca 1980

ctcctgcggt tcctgcggct cggtacggaa gttgaccgtg cttgtctcga tgtagtggtt 2040

gacgatggtg cagaccgccg gcatgtccgc ctcggtggca cggcggatgt cggccgggcg 2100

tcgttctggg ctcatggtag atcccccgtt cgtaaatggt gaaaattttc agaaaattgc 2160

ttttgcttta aaagaaatga tttaaattgc tgcaatagaa gtagaatgct tgattgcttg 2220

agattcgttt gttttgtata tgttgtgttg agaattaatt ctcgaggtcc tctccaaatg 2280

aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt gtgcgtcatc 2340

ccttacgtca gtggagatat cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc 2400

ttctttttcc acgatgctcc tcgtgggtgg gggtccatct ttgggaccac tgtcggtaga 2460

ggcatcttga acgatagcct ttcctttatc gcaatgatgg catttgtagg agccaccttc 2520

cttttccact atcttcacaa taaagtgaca gatagctggg caatggaatc cgaggaggtt 2580

tccggatatt accctttgtt gaaaagtctc aattgccctt tggtcttctg agactgtatc 2640

tttgatattt ttggagtaga caagtgtgtc gtgctccacc atgttatcac atcaatccac 2700

ttgctttgaa gacgtggttg gaacgtcttc tttttccacg atgctcctcg tgggtggggg 2760

tccatctttg ggaccactgt cggcagaggc atcttcaacg atggcctttc ctttatcgca 2820

atgatggcat ttgtaggagc caccttcctt ttccactatc ttcacaataa agtgacagat 2880

agctgggcaa tggaatccga ggaggtttcc ggatattacc ctttgttgaa aagtctcaat 2940

tgccctttgg tcttctgaga ctgtatcttt gatatttttg gagtagacaa gtgtgtcgtg 3000

ctccaccatg ttgacctgca ggcatgcaag cttgcatgcc tgcaggcatg cnagcttgca 3060

tgacctgcag gnnccccaga ttagcctttt caatttcaga aagaatgcta acccacagat 3120

ggttagagag gcttacgcag caggtctcat caagacgatc tacccgagca ataatctcca 3180

ggaaatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca ggactaactg 3240

catcaagaac acagagaaag atatatttct caagatcaga agtactattc cagtatggac 3300

gattcaaggc ttgcttcaca aaccaaggca agtaatagag attggagtct ctaaaaaggt 3360

agttcccact gaatcaaagg ccatggagtc aaagattcaa atagaggacc taacagaact 3420

cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa 3480

aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac 3540

agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct 3600

cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg 3660

tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc 3720

cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 3780

tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 3840

cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 3900

ggagagaaca cgtctagaat gagaaggtgt gggtatagtt tgggccttgg tgagcccaat 3960

ttggacggaa agcccaattt agattacgac gccgtttgtc gtccttctga gcttcacgcg 4020

cttaaaaagg gcgcgttgga ttatattcag aattcggaaa atcagatatt gtttacaatt 4080

catcagattt tcgagtcgtg gattttttcc tcgaaaaaat tgttggatcg aataagtgag 4140

aggatcagta aagaagagtt taccaaagca gcagatgatt gttggatact ggagaaaata 4200

tggaagttat tggaggaaat cgagaattta catttattaa tggatcctga cgatttcctg 4260

catctgaaga cgcaactgag gatgaaaaca gtggcggatt ctgaaacttt ttgttttcga 4320

tcaaaaggac tgatcgaggt aacaaaatta agcaaggatc tacggcacaa ggtgccgaag 4380

atccttggtg tagaggtgga ccctatggga ggaccggtga tacaagagtc ggcaatggag 4440

ttgtaccgag aaaaaagaag atacgagaag atacatctgt tacaagcgtt tcaaggggtg 4500

gaatccgctg ttaaagggtt tttctttaat tataaacagt tgttggtgat catgatgggt 4560

agtttggaag cgaaagcgaa ttttgctgtg attggtggtt ctactgagtc ttcggatttg 4620

ttggctcagt tgtttttaga acctacttat tatccgagtt tggatggtgc caagactttt 4680

attggtgatt gttgggagca tgatcaggct gttggtagcg gcctcgattg tcgtcatcat 4740

cggaagaatc ggactgcgaa acaatgatct agannnggat cccccgaatt tccccgatcg 4800

ttcaaacatt tggcaataaa gtttgtttct taagattgaa tcctgttgcc ggtcttgcga 4860

tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca 4920

tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg 4980

cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta 5040

tgttactaga tcgggaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc 5100

cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 5160

aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 5220

acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 5280

ttggagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt 5340

tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat 5400

atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca 5460

gggttcccct cgggataaat tgggttgttc agttttgggc tggccgaaga aaattaaaag 5520

atagtggagc tcaacccaac taattttctt ctattccact ttaatctaac cctcaattaa 5580

ttagtttaat tttatgacta atttttttaa tgcacataca atgaattcta aaaatttact 5640

tcacacacaa actatcaatt tatataaatt cattatactt atttcaaaag cgaactaatt 5700

ataaactctt tgcattattt caaaagcgaa ctaattataa actctttgca ttattttggt 5760

ttagctagaa atacatgcaa ttccacaaat agttacaaac acagttattc gagtgatgga 5820

tggatggatg tctcaattat ggctgcacaa atacacgagt tgtgccttag ggagaggggg 5880

cgggggaatc atgcttttgg gtggaaaaaa ttcatttcta atgctttgtc tgtttcacct 5940

gaaaagataa ttagtctatt cataagagga gatgag 5976

Claims (7)

1. Cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanked by: the 500bp flanking the left border is as shown in SEQ ID NO 1; the 500bp flanking the right border is as shown in SEQ ID NO. 2.

2. The cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert of claim 1 flanked by: DNA sequence composed of soybean genome derived sequence and exogenous insert derived sequence, as shown in SEQ ID NO. 5.

3. A method for preparing a flank of the exogenous insert of cyst nematode resistant transgenic soybean event ZHs1-2 according to claim 1 or 2, wherein: extracting genome DNA of the transgenic soybean event ZHs1-2, analyzing and determining an insertion site of an exogenous fragment by using a genome re-sequencing technology, and obtaining the transgenic soybean event by PCR amplification.

4. The use of the exogenous insert flanking cyst nematode resistant transgenic soybean event ZHs1-2 according to claim 1 or 2, wherein: and detecting whether the sample to be detected contains the component derived from ZHs 1-2.

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

ZHs1-2 the left border detection primers were:

the forward primer LB-F1 is: f, 5'-ATGCACTATTCAAATAGGAGCA-3', and the method comprises the following steps of,

the reverse primer LB-R1 is: 5'-ATTCAGTACATTAAAAACGTCCG-3', respectively;

when the 821bp segment is obtained by amplification, determining that the component derived from ZHs1-2 is contained in the sample to be detected; otherwise, it does not.

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

ZHs1-2 the right border detection primers were:

the forward primer is: 5'-CCCGATCGTTCAAACATT-3' the flow of the air in the air conditioner,

the reverse primer is: 5'-CTCATCTCCTCTTATGAATAGACTA-3', respectively;

when the 1184bp fragment is obtained by amplification, the component derived from ZHs1-2 is judged to be contained in the sample to be detected. Otherwise, it does not.

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

the sample to be detected comprises plants, tissues, seeds and products corresponding to the parents, derived strains or varieties of ZHs 1-2.

Images