CN108239641B - Disease-resistant transgenic soybean event B5B9104-3 exogenous insert flanking sequence and application thereof - Google Patents
Disease-resistant transgenic soybean event B5B9104-3 exogenous insert flanking sequence and application thereof Download PDFInfo
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Abstract
The invention provides a flanking sequence of an exogenous insert fragment of a disease-resistant transgenic soybean event B5B9104-3 and application thereof, belonging to the technical field of plant biology. In particular to left and right border flanking sequences of a broad-spectrum mosaic virus-resistant transgenic soybean event B5B9104-3 exogenous insertion fragment and application thereof. The left boundary flanking sequence of the exogenous insertion fragment of the transgenic soybean event B5B9104-3 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 insert of the transgenic soybean event B5B9104-3 disclosed by the invention can be used as a target DNA sequence to establish a specific detection method of the transgenic event. The exogenous insert flanking sequence and the detection method provided by the invention are suitable for the specific detection of the transgenic soybean event including parents, derived strains or varieties, and products thereof including plants, tissues, seeds and products.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a flanking sequence of a broad-spectrum mosaic virus-resistant transgenic soybean event B5B9104-3 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 (Rsv1, 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 or segment 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 exogenous SMV-P3 gene RNAi fragments into Shennong No. 9 (national bean 2007015) of a cultivated soybean variety to obtain disease-resistant transgenic soybean B5B 9104-3. 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 B5B9104-3 to soybean mosaic virus No. 3 virulent strain (SMV SC3, major epidemic strain of SMV in northeast soybean producing area) is remarkably higher than that of Shennong No. 9 of a control variety, the Shennong No. 9 shows high resistance, and the resistance can be stably inherited. In addition, B5B9104-3 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 B5B9104-3 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.
By analyzing the existing patents and literatures, the article and patent report related to the flanking sequence of the exogenous insert of the disease-resistant transgenic soybean event B5B9104-3 are not found at present. According to the research, the left and right border flanking sequences of the exogenous insert of the transgenic soybean event B5B9104-3 are obtained by a genome re-sequencing technology and a PCR technology, and the transformation event specificity detection method is established according to the sequence characteristics of the flanking sequences, so that a basis is provided for the commercial application of a broad-spectrum mosaic virus-resistant transgenic soybean event B5B9104-3 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 B5B9104-3 exogenous insertion fragment. The invention also provides a specific detection method of the transgenic event.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
the invention provides a transgenic soybean event B5B9104-3 exogenous insert with left and right border flanking sequences shown in SEQ-2 and SEQ-3, which is characterized in that the transgenic soybean event B5B9104-3 is a DNA sequence consisting of a soybean genome sequence and an exogenous insert sequence. Wherein:
the left border flanking sequence features of the exogenous insert include:
(1) the 1 st to 480 th site sequences of SEQ-2 are derived from the genomic sequence of Shennong No. 9 of a cultivated soybean variety;
(2) the 481-1200 site sequence of SEQ-2 is derived from an exogenous insert sequence.
The characteristics of the right border flanking sequence of the exogenous insert include:
(1) the 1 st-582 th site sequence of SEQ-3 is derived from an exogenous insertion fragment sequence;
(2) the 583-th and 1294-th site sequences of SEQ-3 are derived from the genomic sequence of the cultivated soybean variety Shennong No. 9
The flanking sequences of the left border and the right border of the exogenous insert of the transgenic soybean event B5B9104-3 provided by the invention are obtained by the following steps: (1) the method comprises the steps of taking a disease-resistant transgenic soybean event B5B9104-3 as a material, adopting a genome re-sequencing technology, searching a soybean genome database (http:// soybase. org /), determining that the specific insertion position of an exogenous fragment in a reference soybean genome (Wm82.a2.v1) is 30461895 site of Chr11 chromosome, wherein the insertion mode is forward single-copy insertion, and obtaining sequences of 2kb of the insertion sites at the upstream and downstream 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 a reference soybean genome and the sequence of the insertion fragment, and PCR amplification is carried out by taking the total DNA of the B5B9104-3 genome as a template to obtain the left and right boundary flanking sequences of the exogenous insertion fragment of the transgenic soybean event B5B9104-3 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 preparation detection kit for a transgenic soybean event B5B 9104-3. The method is characterized in that a left boundary flanking sequence of a transgenic soybean event B5B9104-3 exogenous insert is utilized to design a specific detection primer or prepare a specific probe. Wherein one primer is a forward primer designed according to the 1 st to 480 th site sequence of SEQ-2, and the other primer is a reverse primer designed according to the 481 st and 1200 th site sequence of SEQ-2, namely, the two primer combinations are the primers for detecting the specificity of the left boundary flanking sequence of the exogenous insert fragment as described in claim 1.
Preferably, the detection primer specific to the left border flanking sequence of the exogenous insert is:
the forward primer is as follows: 5'-GAAAAAACACATTATTCCATGTGGAGAAG-3' (SEQ-4)
The reverse primer is as follows: 5'-TGAGCAACTTGAAATGATTTCAGAAACTTC-3' (SEQ-5)
The invention provides a specific detection method or a preparation detection kit for a transgenic soybean event B5B 9104-3. 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 9104-3. One primer is a forward primer designed according to the sequence of the 1 st to 582 th sites of SEQ-3, and the other primer is a reverse primer designed according to the sequence of the 583 th and 1294 th sites of SEQ-3, namely, the two primer combinations are the primers for detecting the specificity of the right border flanking sequence of the exogenous insert as claimed 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'-TTCCACACAACATACGAGCCG-3' (SEQ-6)
The reverse primer is as follows: 5'-TGGGGAAAAATACATATTAGAACACTG-3' (SEQ-7)
The invention provides application of flanking sequences of a left boundary and a right boundary of an exogenous insertion segment of a transgenic soybean event B5B9104-3 and a specificity detection method in detection of transgenic soybean event B5B9104-3 including parents, derived strains or varieties, and products thereof including plants, tissues, seeds and products. Designing specific detection primers shown as SEQ-4 and SEQ-5 according to the left boundary flanking sequence of the B5B9104-3 exogenous insert; or designing specific detection primers according to the right border flanking sequence of the B5B9104-3 exogenous insert as shown in SEQ-6 and SEQ-7. DNA samples of roots, stems, leaves, flowers and seeds of transgenic soybean event B5B9104-3 were extracted, respectively, and PCR amplification was performed using Shennong No. 9, a recipient non-transgenic soybean variety, and a conventional soybean variety as controls. The PCR products were separated by electrophoresis on a 1% agarose gel and stained with EB to identify the presence of specifically amplified bands. The length of the left border amplification fragment of the transgenic soybean event B5B9104-3 exogenous insertion fragment is 342 bp. The length of the right border amplification fragment of the transgenic soybean event B5B9104-3 exogenous insertion fragment is 426 bp.
In the invention, the transgenic soybean event B5B9104-3 is obtained by introducing exogenous SMV-P3 gene RNAi fragment into Shennong No. 9 (Cogeneration bean 2007015) of cultivated soybean variety by using agrobacterium-mediated method. The structural map of the B5B9104-3 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, transgenic soybean event B5B9104-3 was publicly available from the agricultural scientific college of Jilin province.
In the invention, the specific detection method of the transgenic soybean event B5B9104-3 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 9104-3.
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 B5B9104-3 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 B5B9104-3, comprises an exogenous insertion fragment sequence and a genome sequence of Shennong No. 9 of a cultivated soybean variety, and determines the specific insertion site of the exogenous fragment in a 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 9104-3.
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 B5B9104-3 including parents, derived strains or varieties and products thereof including plants, tissues, seeds and products, thereby realizing the effective supervision and management of the transgenic soybean and the products thereof.
Drawings
FIG. 1.B5B9104-3 transformation vector pTF101.1-RBSC2-P3i structural map
FIG. 2. transgenic soybean event B5B9104-3 left border flanking sequence specific PCR detection. M: DNA molecular weight standard (DL2000), 1: B5B9104-3, 2: B5B9104-3 stem, 3: B5B9104-3 blade, 4: B5B9104-3 flower, 5: B5B9104-3 seed, 6: soybean variety shennong No. 9, 7: soybean variety Jiyu 47, 8: soybean variety Jiyu 72, 9: corn, 10: cotton
FIG. 3 transgenic Soybean event B5B9104-3 right border flanking sequence specific PCR detection M: DNA molecular weight standard (DL2000), 1: B5B9104-3, 2: B5B9104-3 stem, 3: B5B9104-3 blade, 4: B5B9104-3 flower, 5: B5B9104-3 seed, 6: soybean variety shennong No. 9, 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 exogenous fragment insertion site of transgenic Soybean event B5B9104-3
1. Transgenic soybean B5B9104-3 genome DNA extraction
(1) Extracting genome DNA: taking 1-2g of soybean young leaves, grinding the soybean young leaves into powder by using liquid nitrogen, and filling the powder into a 50mL centrifuge tube. 5mL of extract A (100mmol/L Tris-HCl, pH8.0, 0.35mol/L sorbitol, 5mmol/L EDTA, pH8.0, 1% 2-mercaptoethanol), 3.5mL of extract B (50mmol/L Tris-HCl, pH8.0, 4.0mol/L NaCl, 1.8% CTAB, 25mmol/L EDTA, pH8.0), 0.3mL 30% sodium lauroyl sarcosinate and 2% PVP-360 were added in this order, and incubated at 55 ℃ for 60 to 90 minutes while shaking gently several times. The tube was removed, added with chloroform/isoamyl alcohol (24: 1) of the same volume, shaken gently upside down for 15 minutes, and then centrifuged at room temperature for 10 minutes (13000 rpm). The supernatant was aspirated, 2/3 volumes of pre-cooled isopropanol mixed with 1/10 volumes of sodium acetate in the supernatant were added, and centrifuged at 13000rpm for 20 minutes at 4 ℃. The supernatant was discarded and rinsed with cold 75% ethanol. Air drying the DNA to surface, and storing at-20 deg.C
(2) And (3) genomic DNA purification: with 200uL ddH2O-solubilized DNA, 5uL RNase (10mg/mL) was added, and incubated at 37 ℃ for 40 minutes. Extracted 1-2 times with equal volume of phenol/chloroform and centrifuged at 13000rpm for 10 minutes at room temperature. The supernatant was transferred to a new 1.5mL centrifuge tube and precipitated with an equal volume of pre-cooled 100% chloroform. Centrifuge at 13000rpm for 10 minutes at room temperature. The supernatant was transferred to a new 2mL centrifuge tube and the DNA was precipitated with an amphiploid volume of cold absolute ethanol (1/10 volumes of sodium acetate mixed) and then left at-20 ℃ for 30 minutes. 13000rpm for 15 minutes, rinsing with 75% ethanol for 2 times, and drying in air for 15-20 minutes. 50-100 uL ddH2O dissolves the DNA. DNA concentration was measured by UV spectrophotometer (Quawell Q5000) at-20 deg.CAnd (5) storing for later use.
2. Transgenic soybean B5B9104-3 genome re-sequencing analysis
The transgenic soybean B5B9104-3 was subjected to resequencing 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 42606358 original Reads (paired sequences) were quality assessed and filtered to yield 42038469 clear Reads. Clear Reads were then aligned to the reference genomic sequence (wm82.a2.v1, http:// phytozome. jgi. doe. gov/pz/portal. html # | info ias ═ 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 12.76Gbp, and the Q30 reaches 85.05 percent. The average alignment of the sample to the reference genome was 99.38%, the average depth of coverage was 12X, and the genome coverage was 98.82% (at least one base coverage).
Comparing the transgenic soybean B5B9104-3 genome re-sequencing data with a reference genome and an exogenous insertion sequence respectively, and finding out 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 and the reference genome result by using blastn according to the assembled contig, selecting the contig sequence to be compared to the chromosome region, and carrying out IGV screenshot verification on the bwa comparison result of the regions to obtain the insertion position information of the exogenous insertion fragment. The analysis result shows that the insertion position of the exogenous fragment of the transgenic soybean B5B9104-3 is 30461895 of the chromosome of Chr11, and the insertion mode is positive single-copy insertion. The position of the insertion site in the reference genomic sequence and the-2 kb sequences upstream and downstream thereof (Gm 11: 30460895..30462894) are shown in SEQ-1.
Example 2 analysis of the left and right border flanking sequences of the exogenous insert of transgenic Soybean event B5B9104-3
And designing PCR detection primers according to the exogenous insertion sequence of the transgenic soybean event B5B9104-3 and upstream and downstream sequences of the insertion site in the soybean reference genome. The sequence amplification primers upstream of the insertion site of B5B9104-3 are B5B9104LB-F1 (5'-GCATTATGTTTGAGGGAGACAAGC-3') and B5B9104LB-R1 (5'-AAGAGGGAGGAAGTATGTGGGAG-3'); the sequence amplification primers downstream of the insertion site of B5B9104-3 are B5B9104RB-F1 (5'-CTTTCTTCTGAGTTACATCTTTGTCTG-3') and B5B9104RB-R1 (5'-ATACACAAATGGAGGCTACAACG-3').
PCR amplification was carried out using the above primers, respectively, using the genomic DNA of B5B9104-3 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 B5B9104-3, 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 B5B9104-3 exogenous insert is 1200bp in length, wherein the 1 st to 480 th site sequences are derived from the genome sequence of Shennong No. 9 of a cultivated soybean variety, and the 481 st and 1200 th site sequences are derived from the exogenous insert sequences. The length of the right border flanking sequence (SEQ-3) of the B5B9104-3 exogenous insert is 1294bp, wherein the 1 st-582 th site sequence is derived from the exogenous insert sequence, and the 583 rd and 1294 th site sequences are derived from the genome sequence of the cultivated soybean variety Shennong 9.
Example 3 transgenic Soybean event B5B9104-3 specific PCR assays
Specific detection primers are respectively designed according to the left border flanking sequence (shown as SEQ-2) and the right border flanking sequence (shown as SEQ-3) of the exogenous insert of the transgenic soybean event B5B 9104-3. In the left boundary flanking sequence specificity detection primer combination, one primer is a forward primer designed according to the 1 st to 480 th site sequences of SEQ-2 and is shown as SEQ-4; the other primer is a reverse primer designed according to the 481-1200 th site sequence of SEQ-2 and is shown as SEQ-5. In the primer combination for detecting the specificity of the flanking sequences on the right boundary, one primer is a forward primer designed according to the sequence of the 1 st to 582 th sites of SEQ-3, and is shown as SEQ-6; the other primer is a reverse primer designed according to the 583-th and 1294-th site sequences of SEQ-3, and is shown as SEQ-7.
DNA samples of roots, stems, leaves, flowers and seeds of transgenic soybean plant B5B9104-3 were extracted, respectively, according to the method of example 1. PCR amplification was performed with recipient non-transgenic soybean varieties Shennong No. 9, conventional soybean varieties 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 ddH2O19.5 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 ℃. 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 bands are generated in the non-transgenic soybean varieties Shennong 9, the conventional soybean varieties Jiyu 47 and Jiyu 72 and the corn and the cotton, and only the transgenic soybean B5B9104-3 samples including roots, stems, leaves, flowers and seeds generate specific amplification bands. Wherein the length of the amplified fragment of the left boundary flanking sequence is 342bp, as shown in FIG. 2; the amplified fragment length of the right border flanking sequence is 426bp, as shown in FIG. 3. The research shows that the PCR analysis by using the exogenous insert flanking sequence specific primer can specifically detect whether the sample contains the exogenous insert flanking sequenceB5B 9104-3.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. Broad-spectrum mosaic virus-resistant transgenic soybean event B5B9104-3 exogenous insert left border and right border flanking sequences, and is 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 exogenous insert right border flanking 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 to 480 th site sequences of SEQ-2 are derived from the genomic sequence of Shennong No. 9 of a cultivated soybean variety;
(2) the 481-1200 site sequence of SEQ-2 is derived from an exogenous insertion fragment sequence;
the characteristics of the right border flanking sequence of the exogenous insert include:
(1) the 1 st-582 th site sequence of SEQ-3 is derived from an exogenous insertion fragment sequence;
(2) the 583-th and 1294-th site sequences of SEQ-3 are derived from the genomic sequence of the cultivated soybean variety Shennong No. 9.
2. The method for preparing the flanking sequences of the left and right borders of the exogenous insertion fragment as claimed in claim 1, wherein the genomic DNA of the transgenic soybean event B5B9104-3 is extracted, the exogenous fragment insertion site is determined by analysis of a genome resequencing technique, and the exogenous fragment insertion site is obtained by PCR amplification.
3. Use of the flanking sequences shown in claim 1 for establishing a specific detection method or for preparing a detection kit for transgenic soybean event B5B9104-3, wherein specific primers or probes are prepared based on the flanking sequences shown in claim 1.
4. The use of the flanking sequences of claim 1, the specific detection method or the detection kit of claim 3 for the detection of transgenic soybean event B5B9104-3, wherein the test subject comprises a parent, a derived line or variety, and a product or product thereof.
5. The specific detection method as claimed in claim 3 or claim 4, wherein one primer is a forward primer designed according to the sequence at the 1 st-480 th site of SEQ-2, and the other primer is a reverse primer designed according to the sequence at the 481 st-1200 th site of SEQ-2, i.e. the two primers are combined to be the specific detection primer for the left border flanking sequence of the exogenous insert as claimed in claim 1.
6. The kit as set forth in claim 3 or claim 4, wherein one primer is a forward primer designed according to the sequence at the 1 st-480 th site of SEQ-2, and the other primer is a reverse primer designed according to the sequence at the 481 st-1200 th site of SEQ-2, i.e. the two primers are combined to be the detection primer specific for the left border flanking sequence of the exogenous insert as set forth in claim 1.
7. The primers for detecting the specificity of the flanking sequences of the left border of the exogenous insertion fragment according to claim 5, wherein: as shown in SEQ-4 and SEQ-5:
the forward primer SEQ-4 is: 5'-GAAAAAACACATTATTCCATGTGGAGAAG-3'
The reverse primer SEQ-5 is: 5'-TGAGCAACTTGAAATGATTTCAGAAACTTC-3' are provided.
8. The specific detection method as claimed in claim 3 or claim 4, wherein one primer is a forward primer designed according to the sequence at the 1 st-582 th site of SEQ-3, and the other primer is a reverse primer designed according to the sequence at the 583 nd-1294 th site of SEQ-3, that is, the two primers are combined to be the specific detection primer for the right border flanking sequence of the exogenous insert as claimed in claim 1.
9. The detection kit as claimed in claim 3 or claim 4, wherein one primer is a forward primer designed according to the sequence at the 1 st-582 th site of SEQ-3, and the other primer is a reverse primer designed according to the sequence at the 583 nd-1294 th site of 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 claimed in claim 1.
10. The primer for detecting the specificity of the right border flanking sequence of the exogenous insertion fragment according to claim 8, wherein the primer set forth in SEQ-6 and SEQ-7:
the forward primer SEQ-6 is: 5'-TTCCACACAACATACGAGCCG-3'
The reverse primer SEQ-7 is: 5'-TGGGGAAAAATACATATTAGAACACTG-3' are provided.
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| CN107190003A (en) * | 2017-06-09 | 2017-09-22 | 武汉天问生物科技有限公司 | A kind of method of efficient quick separating T DNA insertion point flanking sequences and application thereof |
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| CN107190003A (en) * | 2017-06-09 | 2017-09-22 | 武汉天问生物科技有限公司 | A kind of method of efficient quick separating T DNA insertion point flanking sequences and application thereof |
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| RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean;Xiangdong Yang等;《Plant Cell Rep》;20170729;第37卷(第1期);第103-114页 * |
| RNAi介导SMV-P3 基因沉默增强大豆对花叶病毒病的抗性;杨向东 等;《作物学报》;20161110;第42卷(第11期);第1647-1655页 * |
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