CN108239637B - Disease-resistant transgenic soybean event B5B8127-3 exogenous insert flanking sequence and application thereof - Google Patents
Disease-resistant transgenic soybean event B5B8127-3 exogenous insert flanking sequence and application thereof Download PDFInfo
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Abstract
The invention provides a disease-resistant transgenic soybean event B5B8127-3 exogenous insert flanking sequence and application thereof, belonging to the technical field of plant biology. In particular to left and right border flanking sequences of a broad-spectrum anti-mosaic virus transgenic soybean event B5B8127-3 exogenous insertion fragment and application thereof. The left boundary flanking sequence of the exogenous insertion fragment of the transgenic soybean event B5B8127-3 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 B5B8127-3 disclosed by the invention can be used as a target DNA sequence, and a specific detection method of the transgenic event is established. 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 broad-spectrum mosaic virus-resistant transgenic soybean event B5B8127-3 exogenous insert and application thereof.
Background
Potyvirus (Potyvirus) is the largest genus of plant viruses, including about 200 established species and tentative species. The virus can infect various plants of Solanaceae, Chenopodiaceae, Leguminosae, Cucurbitaceae and the like, and cause serious yield loss. Soybean Mosaic Virus (SMV), Bean Common Mosaic Virus (BCMV), and Watermelon Mosaic Virus (WMV) belong to the genus potyvirus. All three viruses can be transmitted by seed borne virus or aphid, and cause symptoms such as mosaic, leaf curl, plant dwarfing and the like (Gao et al.2015; Yang et al.2014). SMV is a main viral disease affecting soybean production and is one of the most important diseases in each main soybean production area in China. SMV generally causes soybean yield reduction of 10-35%, and even large-area dead birth in severe years and regions (Yang et al 2013, 2014; Ross 1983). Infection with SMV often causes mottle soybean kernels, seriously affecting the quality of appearance and commodity value of the kernels. The foreign division of SMV strains into G1-G7(Cho and Goodman 1979) by their pathogenic response to the identified host, and the division of SMV strains into SC1-SC22 in China (Li et al 2010; Wang et al 2003). Because the chemical prevention and control are difficult and the environmental safety is easy to cause, the prevention and control of the soybean mosaic virus in production mainly depend on the cultivation of an SMV resistant soybean variety. The other 2 viruses, BCMV and WMV, although not severe in soybean production, still have a major potential hazard to soybean production (Zhou et al 2014), particularly the synergistic interaction between BCMV, WMV and SMV, often causing variation in SMV strains and more severe yield losses (Anjos, et al 1992; Reddy, et al 2001). Currently, soybean varieties carrying SMV resistance sites (Rsvl, Rsv3, Rsv4) are mainly used in production to control SMV damage (Yu, 1994; Hayes, et al., 2000; Gore, et al., 2002; Jeong and Maroof, 2008; Maroof, et al., 2010). But gradual loss of the original resistance of resistant varieties results from forward screening pressure caused by extensive planting of resistant varieties, SMV genomic variation, and interaction between SMV and hosts or other infecting soybean viruses (Koo, et al, 2005; Choi, et al, 2005; Gagarinova, et al, 2008). Some SMV races have been reported to be resistant to commercial soybean varieties carrying the resistance genes Rsv1, Rsv3, and Rsv4 (Choi, et al, 2005). The wide variation of SMV and the mixed infection of multiple SMV strains or other viruses such as BCMV make the control of soybean viral diseases increasingly difficult.
The existing research shows that the dsRNA produced by expressing partial virus genome sequence segments in plants can effectively inhibit the infection of viruses. Wang et al (2001) introduced the coat protein gene CP carrying the SMV 3' -UTR into soybean, in which 2 transgenic lines had significantly improved levels of resistance to SMV compared to the recipient variety. Furutani et al (2006) introduced the SMV-CP gene into soybean, and the results of the inoculation and identification showed that the transgenic soybean plants had a higher level of resistance to SMV infection. Zhang et al (2011) and Kim et al (2013) introduce an SMV-CP gene sequence Inverted Repeat (IR) into soybeans, and the inoculation identification result shows that the transgenic soybeans can effectively resist infection of SMV, and the SMV resistant level of the transgenic soybeans is obviously higher than that of receptor varieties. Recently, Gao et al (2015) introduced an IR fragment of SMV HC-Pro gene, a negative regulator involved in post-transcriptional gene silencing (PTGS), into soybeans, and vaccination identification showed that transgenic soybeans were significantly improved in SMV resistance. Therefore, RNAi silencing of the SMV coding gene mediated by the host is an effective means for improving the SMV resistance of the soybean, and more importantly, an effective technical approach is provided for simultaneously resisting various viruses and different physiological races of the viruses by using an RNAi interference technology.
The Jilin province academy of agricultural sciences adopts an agrobacterium-mediated method to introduce an exogenous SMV-P3 gene RNAi fragment into a cultivated soybean variety Williams82 to obtain a disease-resistant transgenic soybean B5B 8127-3. The size of the RNAi fragment of the SMV-P3 gene is 302bp, the RNAi fragment is positioned at 2529-2834nt sites of an SMV genome, and the promoter is a kidney bean leaf specific promoter RBSC 2. The resistance identification result shows that the resistance level of B5B8127-3 to soybean mosaic virus No. 3 virulent strain (SMV SC3, a main epidemic strain of SMV in northeast soybean producing area) is remarkably higher than that of a control variety Williams82, the resistance is high, and the resistance can be stably inherited. In addition, B5B8127-3 shows strong broad-spectrum resistance to 7 main virus types or small varieties including SMV SC3, SMV SC7, SMV SC15, SMV SC18, SMV-R, BCMV and WMV in the soybean main producing area in China. At present, the transgenic soybean event B5B8127-3 enters a safety evaluation stage, and with the promotion of important special items for breeding new national transgenic organism varieties, 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, an article and a patent report related to a flanking sequence of the exogenous insert of the disease-resistant transgenic soybean event B5B8127-3 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 B5B8127-3 are obtained through 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 a broad-spectrum mosaic virus resistant transgenic soybean event B5B8127-3 and derived varieties or strains thereof. On the basis, the invention is provided.
Disclosure of Invention
The invention aims to provide left and right border flanking sequences of a broad-spectrum mosaic virus-resistant transgenic soybean event B5B8127-3 exogenous insertion fragment. The invention also provides a specific detection method of the transgenic event.
The invention is realized by the following technical scheme:
the invention provides a transgenic soybean event B5B8127-3 exogenous insert with left and right border flanking sequences shown in SEQ-2 and SEQ-3, which is characterized in that the sequence is a DNA sequence consisting of a soybean genome-derived sequence and an exogenous insert-derived sequence. Wherein:
the left border flanking sequence features of the exogenous insert include:
(1) the 1 st-320 th site sequence of SEQ-2 is derived from the genome sequence of cultivated soybean Williams 82;
(2) the 321-943 site sequence of SEQ-2 is derived from the exogenous insertion fragment sequence.
The characteristics of the right border flanking sequence of the exogenous insert include:
(1) the 1 st-377 site sequence of SEQ-3 comes from the sequence of the exogenous insertion fragment;
(2) the 378-887 site sequence of SEQ-3 is derived from the genome sequence of cultivated soybean Williams 82.
The flanking sequences of the left boundary and the right boundary of the insertion site of the exogenous fragment of the transgenic soybean event B5B8127-3 are obtained by the following steps: (1) using disease-resistant transgenic soybean event B5B8127-3 as material, adopting genome re-sequencing technology, and searching soybean genome database (http：//soybase.org/) Determining the specific insertion position of the exogenous fragment in a reference soybean genome (Wm82.a2.v1) to be 10447906 site of Chr07 chromosome, wherein the insertion mode is reverse single-copy insertion, and obtaining sequences of upstream and downstream-2 kb of the insertion site in the reference soybean genome, as shown in SEQ-1. (2) Primers are designed according to the upstream and downstream sequences of the insertion position of the exogenous fragment in the reference soybean genome and the sequence of the insertion fragment, and PCR amplification is carried out by taking the total DNA of the B5B8127-3 genome as a template to obtain the left and right boundary flanking sequences of the exogenous insertion fragment of the transgenic soybean event B5B8127-3 as shown in 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 invention provides a specific detection method or a detection kit for a transgenic soybean event B5B 8127-3. The method is characterized in that a left boundary flanking sequence of a transgenic soybean event B5B8127-3 exogenous insertion fragment is utilized to design a specific detection primer or prepare a specific probe. Wherein one primer is a forward primer designed according to the sequence of the 1 st-320 th site of SEQ-2, and the other primer is a reverse primer designed according to the sequence of the 321. sup. 943 rd site of SEQ-2, i.e., the two primer combinations are the primers for detecting the specificity of the left border flanking sequence of the exogenous insert as claimed 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'-TGAGAGGAGAGGGAAACGAGA-3' (SEQ-4)
The reverse primer is as follows: 5'-GCTGGCGTAATAGCGAAGAG-3' (SEQ-5)
The invention provides a specific detection method or a detection kit for a transgenic soybean event B5B 8127-3. The method 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 insert of the transgenic soybean event B5B 8127-3. One primer is a forward primer designed according to the sequence at the 1 st-377 th site of SEQ-3, and the other primer is a reverse primer designed according to the sequence at the 378-887 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'-TCTGAGTTACATCTTTGTCTGGTTGTAATG-3' (SEQ-6)
The reverse primer is as follows: 5'-AGTAATAGTGCTATGTACCATATGACAT-3' (SEQ-7)
The invention provides a transgenic soybean event B5B8127-3 exogenous insertion fragment left boundary and right boundary flanking sequence and a specific detection method, and application thereof in detection of transgenic soybean event B5B8127-3 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 B5B8127-3 exogenous insert; or designing specific detection primers according to the right border flanking sequence of the B5B8127-3 exogenous insert as shown in SEQ-6 and SEQ-7. Transgenic soybean event B5B8127-3 root, stem, leaf, flower and seed DNA samples were extracted separately and PCR amplified 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 left border amplification fragment length of the transgenic soybean event B5B8127-3 exogenous insertion fragment is 397 bp. The length of the right border amplification fragment of the transgenic soybean event B5B8127-3 exogenous insertion fragment is 411 bp.
In the invention, the transgenic soybean event B5B8127-3 is obtained by introducing an exogenous SMV-P3 gene RNAi fragment into a cultivated soybean variety Williams82 by utilizing an agrobacterium-mediated method. The structural map of the B5B8127-3 transformation vector pTF101.1-RBSC2-P3i is shown in figure 1. The vector carries an RNAi fragment expression frame of the SMV-P3 gene and a screening marker gene BAR expression frame. The transformation vector pTF101.1-RBSC2-P3i and transgenic soybean event B5B8127-3 were publicly available from the agricultural scientific college of Jilin province.
In the invention, the specific detection method of the transgenic soybean event B5B8127-3 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 45 s; 5min at 72 ℃. Detecting whether a specific band exists in the PCR amplification product by using 1% agarose gel electrophoresis, and analyzing whether the sample contains components derived from B5B 8127-3.
The invention has the beneficial effects that:
1. the invention discloses flanking sequences of a left boundary and a right boundary of a broad-spectrum mosaic virus-resistant transgenic soybean event B5B8127-3 exogenous insertion fragment for the first time.
2. The invention firstly analyzes and confirms the composition of the left and right boundary flanking sequences of the exogenous insertion fragment of the transgenic soybean event B5B8127-3, comprises the sequence of the exogenous insertion fragment and the genome sequence of a cultivated soybean variety Williams82, 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 insert, and establishes a specific qualitative PCR detection method or a preparation detection kit for the transgenic soybean event B5B 8127-3.
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 B5B8127-3 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.
Drawings
FIG. 1.B5B8127-3 transformation vector pTF101.1-RBSC2-P3i structural map
FIG. 2. transgenic soybean event B5B8127-3 left border flanking sequence specific PCR detection. M: DNA molecular weight standard (DL2000), 1: B5B8127-3, 2: B5B8127-3 stem, 3: B5B8127-3 leaves, 4: B5B8127-3 flower, 5: B5B8127-3 seed, 6: soybean variety Williams82, 7: soybean variety Jiyu 47, 8: soybean variety Jiyu 72, 9: corn leaf, 10: cotton leaf
FIG. 3 transgenic Soybean event B5B8127-3 Right boundary flanking sequence specific PCR detection M: DNA molecular weight standard (DL2000), 1: B5B8127-3, 2: B5B8127-3 stem, 3: B5B8127-3 leaves, 4: B5B8127-3 flower, 5: B5B8127-3 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 analysis of insertion sites of exogenous fragments of transgenic Soybean event B5B8127-3
1. Transgenic soybean B5B8127-3 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 of LTris-HCl, pH8.0, 0.35mol/L of sorbitol, 5mmol/L of EDTA, pH8.0, 1% of 2-mercaptoethanol), 3.5mL of extract B (50mmol/L of Tris-HCl, pH8.0, 4.0mol/L of NaCl, 1.8% of CTAB, 25mmol/L of EDTA, pH8.0), 0.3mL of 30% of sodium lauroyl sarcosinate and 2% of PVP-360 were added in this order, and incubated at 55 ℃ for 60 to 90 minutes with gentle shaking 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 B5B8127-3 genome re-sequencing analysis
The transgenic soybean B5B8127-3 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 48643244 original Reads (paired sequences) were quality assessed and filtered to yield 47497008 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 the analysis is clear Data of 14.57Gbp, and Q30 reaches 85.02%. The average alignment of the sample to the reference genome was 99.37%, the average depth of coverage was 13X, and the genome coverage was 98.82% (at least one base coverage).
Comparing the re-sequencing data of the transgenic soybean B5B8127-3 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. The analysis result shows that the insertion site of the exogenous fragment of the transgenic soybean B5B8127-3 is 10447906 site of the chromosome of Chr07, and the insertion mode is reverse single-copy insertion. The position of the insertion site in the reference genomic sequence and the-2 kb sequences upstream and downstream thereof (Gm 07: 10448906..10446905) are shown in SEQ-1.
Example 2 analysis of the left and right border flanking sequences of the exogenous insert of transgenic Soybean event B5B8127-3
And designing PCR detection primers according to the exogenous insertion sequence of the transgenic soybean event B5B8127-3 and the upstream and downstream sequences of the insertion site in the soybean reference genome. B5B8127-3 insertion site upstream sequence amplification primers are B5B8127LB-F1 (5'-AAAGTCAAAGAAGGCGAGGATTGC-3') and B5B8127LB-R1 (5'-GGGTAAGTCACCTAAGACACTCCA-3'); the downstream sequence amplification primers of the B5B8127-3 insertion site are B5B8127RB-F1 (5'-TCTGAGTTACATCTTTGTCTGGTTGTAATG-3') and B5B8127RB-R1 (5'-CATTATGATTTTAATTAATAAATCGTTGTTCAGT-3').
PCR amplification was carried out using the above primers, respectively, using the genomic DNA of B5B8127-3 as a template. 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 left boundary flanking sequence of the exogenous insertion fragment of the transgenic soybean event B5B8127-3, which is shown as SEQ-2, and a right boundary flanking sequence which is shown as SEQ-3. The sequence is composed of DNA sequence from soybean genome sequence and exogenous insertion fragment sequence.
The left border flanking sequence (SEQ-2) of the B5B8127-3 exogenous insert is 943bp in length, wherein the 1 st-320 th site sequence is derived from the genome sequence of cultivated soybean Williams82, and the 321 st-943 rd site sequence is derived from the sequence of the exogenous insert. The length of the right border flanking sequence (SEQ-3) of the B5B8127-3 exogenous insertion fragment is 887bp, wherein the 1 st-377 site sequence is derived from the exogenous insertion fragment sequence, and the 378-887 th site sequence is derived from the genome sequence of the cultivated soybean Williams 82.
Example 3 transgenic Soybean event B5B8127-3 specific PCR assays
Specific detection primers are respectively designed according to the left boundary flanking sequence (shown as SEQ-2) and the right boundary flanking sequence (shown as SEQ-3) of the exogenous insert of the transgenic soybean event B5B 8127-3. In the left boundary flanking sequence specificity detection primer combination, one primer is a forward primer designed according to the sequence of the 1 st to 320 th sites of SEQ-2, and is shown as SEQ-4; the other primer is a reverse primer designed according to the 321-943 site sequence of SEQ-2 and is shown as SEQ-5. In the primer combination for detecting the specificity of the flanking sequence of the right boundary, one primer is a forward primer designed according to the sequence of the 1 st to 377 th sites of SEQ-3, and is shown as SEQ-6; the other primer is a reverse primer designed according to the 378-887 site sequence of SEQ-3, and is shown as SEQ-7.
DNA samples of roots, stems, leaves, flowers and seeds of transgenic soybean plants B5B8127-3 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 45 s; 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, no amplification band exists in the non-transgenic soybean variety Williams82, the conventional soybean variety Jiyu 47 and Jiyu 72 and the corn and cotton, and only the transgenic soybean B5B8127-3 sample including 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 397bp, as shown in FIG. 2; the amplified fragment of right border flanking sequence is 411bp in length, as shown in FIG. 3. The research shows that PCR analysis with exogenous inserted fragment flanking sequence specific primer can detect whether the sample contains component from B5B8127-3 specifically.
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 (7)
1. The broad-spectrum mosaic virus-resistant transgenic soybean event B5B8127-3 exogenous insert left 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, and the sequence is a DNA sequence consisting of a soybean genome-derived sequence and an exogenous insert-derived sequence; wherein:
the left border flanking sequence features of the exogenous insert include:
(1) the 1 st-320 th site sequence of SEQ-2 is derived from the genome sequence of cultivated soybean Williams 82;
(2) the 321-943 site sequence of SEQ-2 comes from the exogenous insertion fragment sequence;
the characteristics of the right border flanking sequence of the exogenous insert include:
(1) the 1 st-377 site sequence of SEQ-3 comes from the sequence of the exogenous insertion fragment;
(2) the 378-887 site sequence of SEQ-3 is derived from the genome sequence of cultivated soybean Williams 82.
2. Use of the flanking sequences shown in claim 1 for establishing a specific detection method or preparing a detection kit for transgenic soybean protein B5B8127-3, wherein specific primers or probes are prepared according to the flanking sequences shown in claim 1.
3. Use of the flanking sequences of claim 1 or the specific detection method or the detection kit of claim 2 for the detection of transgenic soybean event B5B8127-3, wherein the test subject comprises a parent, a derived line or variety, and a product or product thereof.
4. The use as claimed in claim 2 or claim 3, wherein one primer is a forward primer designed according to the sequence at position 1-320 of SEQ-2 and the other primer is a reverse primer designed according to the sequence at position 321-943 of SEQ-2, i.e. the two primers are combined to be a detection primer specific for the left border flanking sequence of the exogenous insert as described in claim 1.
5. The primers for detecting the specificity of the flanking sequences of the left border of the exogenous insertion fragment according to claim 4, wherein: as shown in SEQ-4 and SEQ-5:
the forward primer SEQ-4 is: 5'-TGAGAGGAGAGGGAAACGAGA-3'
The reverse primer SEQ-5 is: 5'-GCTGGCGTAATAGCGAAGAG-3' are provided.
6. The use as claimed in claim 2 or claim 3, wherein one primer is a forward primer designed according to the sequence at positions 1-377 of the sequence shown in SEQ-3, and the other primer is a reverse primer designed according to the sequence at positions 378-887 of the sequence shown in SEQ-3, i.e. the two primers are combined to be the detection primer specific for the right border flanking sequence of the exogenous insert as described in claim 1.
7. The primer for detecting the specificity of the right border flanking sequence of the exogenous insertion fragment according to claim 6, wherein the primer set forth in SEQ-6 and SEQ-7:
the forward primer SEQ-6 is: 5'-TCTGAGTTACATCTTTGTCTGGTTGTAATG-3'
The reverse primer SEQ-7 is: 5'-AGTAATAGTGCTATGTACCATATGACAT-3' are provided.
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