CN108239638A - Disease-resistant transgenic soybean event B5B9013-4 external source Insert Fragment flanking sequences and its application - Google Patents
Disease-resistant transgenic soybean event B5B9013-4 external source Insert Fragment flanking sequences and its application Download PDFInfo
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Abstract
The present invention provides a kind of 4 external source Insert Fragment flanking sequences of disease-resistant transgenic soybean event B5B9013 and its application, belongs to plant biotechnology field.In particular it relates to a kind of left and right boundary flanking sequence of 4 external source Insert Fragments of wide spectrum mosaic disease resisting poison transgenic soybean event B5B9013 and its application.4 external source Insert Fragment left margin flanking sequences of transgenic soybean event B5B9013 disclosed by the invention are as shown in SEQ 2, and right margin flanking sequence is as shown in SEQ 3.4 external source Insert Fragment flanking sequences of transgenic soybean event B5B9013 disclosed by the invention may be used as target dna sequence, establish the transgenic event method for detecting specificity.External source Insert Fragment flanking sequence provided by the invention and detection method are suitable for including the transgenic soybean event parent, derivative strain or kind and its product includes the specific detection of plant, tissue, seed and product.
Description
Technical field
The present invention relates to plant biotechnology fields, and in particular, to a kind of wide spectrum mosaic disease resisting poison genetically engineered soybean thing
Part B5B9013-4 external source Insert Fragment flanking sequences and its application.
Background technology
Potyvirus (Potyvirus) is a maximum category in plant virus, determined kind including about 200 kinds and
Tentative species.The viroid can infect the various plants such as Solanaceae, Chenopodiaceae, pulse family, Curcurbitaceae, and cause serious production loss.
Soybean mosaic virus (soybean mosaic virus, SMV), Bean common mosaic virus (bean common mosaic
Virus, BCMV) and watermelon mosaic virus (watermelon mosaic virus, WMV) belong to Potyvirus.Three
Kind virus can be propagated by Seed transmission or aphid, and cause the symptoms (Gao such as floral leaf, leaf rolling, plant dwarfing
et al.2015；Yang et al.2014).SMV is each soybean of main Disease and China for influencing Soybean production
One of most important disease in main producing region.SMV can generally cause the soybean underproduction 10%~35%, and serious time and area are even caused
Large area total crop failure (Yang et al.2013,2014；Ross 1983).It is mottled that the infecting of SMV typically results in soybean kernel, seriously
Influence the exterior quality and commodity value of seed.SMV strains isolations are by foreign countries to the pathogenic reaction for identifying host by SMV
SMV strains isolations are SC1-SC22 (Li et al.2010 by G1-G7 (Cho and Goodman 1979), China；Wang et
al.2003).Since chemical prevention is difficult and the problems such as easily causing Environmental security, to the prevention master of soybean mosaic virus in production
To depend on the cultivation of SMV resistance soybean varieties.Although a situation arises in Soybean production by other 2 kinds of virus-BCMV and WMV not
Seriously, but to Soybean production still have larger potential hazard (Zhou et al.2014), particularly BCMV, WMV and SMV it
Between collaboration interaction, often result in the variation of SMV strains and even more serious production loss (Anjos, et al.1992；
Reddy, et al.2001).Production at present is upper mainly to utilize the soybean varieties for carrying SMV resistance locus (Rsv1, Rsv3, Rsv4)
Control SMV harm (Yu, 1994；Hayes, et al., 2000；Gore, et al., 2002；Jeong and Maroof,
2008；Maroof, et al., 2010).But due to screening pressure positive caused by the extensive plantation of resistant variety, SMV genomes
Variation and SMV and host or other infect interaction between Viruses Infecting Soybean Plant, cause the gradual forfeiture of the original resistance of disease-resistant variety
(Koo, et al., 2005；Choi, et al., 2005；Gagarinova, et al., 2008).It has been reported that and shows part SMV
Microspecies to carry Rsv1, Rsv3 and Rsv4 resistant gene commercialization soybean varieties generate resistance (Choi, et al.,
2005).The mixed infection of the extensive variation of SMV and multiple SMV strains or other viruses such as BCMV cause Viruses Infecting Soybean Plant venereal disease to do harm to
Prevention is further difficult.
Existing research shows that expressing fractionated viral genomic sequence fragment in plant, the dsRNA of generation can be effective
Inhibit infecting for virus.The coat protein gene CP that Wang etc. (2001) will carry 3 '-UTR of SMV imports soybean, wherein 2 turn
Gene strain significantly improves SMV resistance levels compared with receptor kind.Furutani etc. (2006) by SMV-CP Gene Into Soybeans,
Inoculated identification the result shows that, Transgenic soybean plants infect SMV with higher resistance level.Zhang etc. (2011) and Kim
Deng (2013) by SMV-CP gene orders inverted repeat (inverted repeat, IR) import soybean, inoculated identification result
Show that genetically engineered soybean can effectively resist infecting for SMV, SMV resistance level is significantly higher than receptor kind.Recently, Gao etc.
(2015) negative regulation of posttranscriptional gene silencing (post-transcriptional gene silencing, PTGS) will be participated in
Factor S MV HC-Pro gene IR segments import soybean, and inoculated identification shows that genetically engineered soybean significantly improves SMV resistances.It can
See, it is to improve the effective means of Soybean Resistance SMV to mediate SMV encoding gene RNAi silences using host, it is even more important that is utilized
RNAi perturbation techniques provide a more efficiently technological approaches for a variety of viruses anti-simultaneously and the different biological strains of virus.
External source SMV-P3 gene RNAis segment is imported the cultivated soybean by Jilin Academy of Agricultural Science using agrobacterium-mediated transformation
Kind Shen Nong No. 9 (state examines beans 2007015) obtains disease-resistant transgenic soybean B5B9013-4.SMV-P3 gene RNAi clip sizes
For 302bp, positioned at SMV genome 2529-2834nt sites, promoter is Phaseolus Leaves specificity promoter RBSC2.Resistance is reflected
The result shows that, B5B9013-4 is to No. 3 virulent strain departments of soybean mosaic virus (SMV SC3, northeast soybean producing region SMV Major Epidemics calmly
Strain) disease index be 4.44-13.88, pole is substantially less than check variety Shen Nong 9 (disease index 39.80-53.36, middle sense
Or susceptible), highly resistance is shown as, and resistance can stablize heredity.In addition, B5B9013-4 is to 7 kinds of main diseases of China's major soybean production areas
Malicious type or microspecies show as relatively strong including SMV SC3, SMV SC7, SMV SC15, SMV SC18, SMV-R, BCMV and WMV
Resistance of wide spectrum.Transgenic soybean event B5B9013-4 has been enter into the safety evaluation stage at present, as national genetically modified organism is new
The propulsion of breed of variety key special subjects, the transformation event and its derived varieties or strain are expected to enter commercial applications.
It is to realize that genetically modified plants effectively supervise pipe to transgenic event and its derivative strain or varietY specificity detection
Reason ensures the important technical that transgenosis industry develops in a healthy way.The flanking sequence of external source Insert Fragment and according to this flank sequence
The detection method established is arranged, is the important evidence that genetically modified plants and products thereof are carried out with effective supervision and management.At present
Through thering is related patents and document report genetically modified plants external source to be inserted into flanking sequence.Magnify (2006) such as soldiers and utilize TAIL-PCR
Method analyzes the flanking sequence of the external source Insert Fragment of corn strain MON863, establishes the strain of transgenosis MON863 corns
Method for detecting specificity.Xie Jiajian etc. (2007) is obtained using the methods of TAIL-PCR, genome walking and LD-PCR
The profit such as the flanking sequence of the external source Insert Fragment of transgenic paddy rice Kemingdao, Bt Shans excellent 863 and Ke Feng 6, Yang Zhengyou (2012)
The flanking sequence of the external source Insert Fragment of transgenic rice lines SK-2 is established with TAIL-PCR methods, and establishes the strain
The detection method of specificity.
Existing patent and document are analyzed, not yet find disease-resistant transgenic soybean event B5B9013-4 external sources at present
The relevant article of Insert Fragment flanking sequence and patent report.This research is obtained by genome weight sequencing technologies and round pcr
The left and right boundary flanking sequence of transgenic soybean event B5B9013-4 external source Insert Fragments, and according to its sequence signature, establishing should
Transformation event method for detecting specificity, be wide spectrum mosaic disease resisting poison transgenic soybean event B5B9013-4 and its derived varieties or
Strain commercial applications provide foundation.On this basis, the present invention is proposed.
Invention content
The purpose of the present invention is to provide wide spectrum mosaic disease resisting poison transgenic soybean event B5B9013-4 external source Insert Fragments
Left and right boundary flanking sequence.The present invention also provides the transgenic event method for detecting specificity.
The present invention is achieved through the following technical solutions：
The present invention provides transgenic soybean event B5B9013-4 external source Insert Fragments left and right boundary flanking sequence such as SEQ-2
Shown in SEQ-3, which is characterized in that origin is derived from soybean genomic sequence and from external source Insert Fragment sequence composition
DNA sequence dna.Wherein：
External source Insert Fragment left margin flanking sequence feature includes：
(1) SEQ-2 1-1071 site sequences derive from No. 9 genome sequences of the cultivated soybean Shen Nong；
(2) SEQ-2 1072-1401 site sequences derive from external source Insert Fragment sequence.
External source Insert Fragment right margin flanking sequence feature includes：
(1) SEQ-3 1-323 site sequences derive from external source Insert Fragment sequence；
(2) SEQ-3 324-845 site sequences derive from No. 9 genome sequences of the cultivated soybean Shen Nong.
Transgenic soybean event B5B9013-4 external source Insert Fragment left margins and right margin flanking sequence provided by the invention
It obtains in the following way：(1) using disease-resistant transgenic soybean event B5B9013-4 as material, sequencing technologies are weighed using genome,
And retrieve soybean genome database (http://soybase.org/), determine exogenous sequences with reference to soybean genome
(Wm82.a2.v1) the specific insertion position in is 46747946 site of Chr04 chromosomes, and inserted mode is double copy unit points
It is inserted into, and obtains insertion point and referring to soybean genome middle and upper reaches and downstream~2kb sequences, as shown in SEQ-1.(2) basis
With reference to exogenous sequences insertion position upstream, downstream sequence and Insert Fragment primers in soybean genome, with
B5B9013-4 genome DNAs carry out PCR amplification for template, obtain transgenic soybean event B5B9013-4 external source Insert Fragments
Left and right boundary flanking sequence is as shown in SEQ-2 and SEQ-3.The sequence origin is derived from soybean genomic sequence and from outer
The DNA sequence dna of source Insert Fragment sequence composition.
In view of the integration of exogenous sequences in the plant genome has the characteristics that randomness in transgenic event, difference turns base
Because the insertion point of event its exogenous sequences in genome is different.For specific transgenic event, flanking sequence
It is special.Therefore, specific detection can be carried out to transgenic event using Insert Fragment flanking sequence.Portion is included as utilized
Flanking sequence and the probe of partial exogenous Insert Fragment sequence is divided to be hybridized or designed comprising partial flanking sequences and part
The specific primer of external source Insert Fragment sequence carries out PCR amplification etc..
The present invention provides transgenic soybean event B5B9013-4 method for detecting specificity or detection kit.Its feature exists
In, using transgenic soybean event B5B9013-4 external source Insert Fragment left margin flanking sequences, design specific detection primer or
Prepare specific probe.Wherein one article of primer is the forward primer according to the design of SEQ-2 1-1071 site sequences, and another is drawn
Object is the reverse primer according to the design of SEQ-2 1072-1401 site sequences, i.e., described two primers are combined as claims
The 1 external source Insert Fragment left margin flanking sequence specific detection primer.
Preferably, the external source Insert Fragment left margin flanking sequence specific detection primer is：
The forward primer is：5’-ACGCAAGCAGATACTTCACCT-3’(SEQ-4)
The reverse primer is：5’-TCAGATTGTCGTTTCCCGCC-3’(SEQ-5)
The present invention provides transgenic soybean event B5B9013-4 method for detecting specificity or detection kit.Its feature exists
In, using transgenic soybean event B5B9013-4 external source Insert Fragment right margin flanking sequences, design specific detection primer or
Prepare specific probe.Wherein one article of primer is the forward primer according to the design of SEQ-3 1-323 site sequences, and another is drawn
Object is the reverse primer according to the design of SEQ-3 324-845 site sequences, i.e., described two primers are combined as claims 1
The external source Insert Fragment right margin flanking sequence specific detection primer.
Preferably, the external source Insert Fragment right margin flanking sequence specific detection primer is：
The forward primer is：5’-TCCACACAACATACGAGCCG-3’(SEQ-6)
The reverse primer is：5’-GCATACATCACATTCACACCCA-3’(SEQ-7)
The present invention provide transgenic soybean event B5B9013-4 external source Insert Fragment left margins and right margin flanking sequence and
Method for detecting specificity includes parent, derivative strain or kind in transgenic soybean event B5B9013-4 and its product includes planting
Application in strain, tissue, seed and product testing.It is designed according to B5B9013-4 external source Insert Fragment left margins flanking sequence special
Different in nature detection primer is as shown in SEQ-4 and SEQ-5；Or it is designed according to B5B9013-4 external source Insert Fragment right margins flanking sequence
Specific detection primer is as shown in SEQ-6 and SEQ-7.Extraction transgenic soybean event B5B9013-4 roots, stem, leaf, Hua He respectively
Seed DNA sample, and using receptor Non-transgenic soybean kind Shen Nong 9 and conventional soy kind as control, carry out PCR expansions
Increase.PCR product is detached through 1% agarose gel electrophoresis, and is dyed using EB, with identification with the presence or absence of specific amplification item
Band.The transgenic soybean event B5B9013-4 external sources Insert Fragment left margin expanding fragment length is 355bp.It is described to turn base
Because soybean event B5B9013-4 external source Insert Fragment right margins expanding fragment length is 316bp.
In the present invention, transgenic soybean event B5B9013-4 is to utilize agrobacterium-mediated transformation by external source SMV-P3 genes
RNAi segments import what the cultivated soybean kind Shen Nong 9 (state examines beans 2007015) was obtained.B5B9013-4 conversion carriers
PTF101.1-RBSC2-P3i structure collection of illustrative plates is as shown in Figure 1.The carrier carries SMV-P3 gene RNAi fragment expression frames and sieve
Select marker gene BAR expression cassettes.The conversion carrier pTF101.1-RBSC2-P3i and transgenic soybean event B5B9013-4 is public
Crowd can obtain from Jilin Academy of Agricultural Science.
In the present invention, the transgenic soybean event B5B9013-4 method for detecting specificity, PCR reaction systems (25uL)
For：10 × PCR buffer solutions 2.5uL, 10mmol/L dNTPs 0.5uL, 5U/uL Taq enzyme 0.5uL, DNA sample 1.0uL,
10umol/L forward primers 0.5uL, 10umol/L reverse primer 0.5uL, ddH2O 19.5uL.PCR reaction conditions are：95℃
5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 45s, totally 30 recycle；72℃ 5min.It is examined using 1% agarose gel electrophoresis
It surveys in pcr amplification product with the presence or absence of specific band, analyzes the ingredient whether contained in sample from B5B9013-4.
Beneficial effects of the present invention
1. the first public wide spectrum mosaic disease resisting poison transgenic soybean event B5B9013-4 external source Insert Fragments left side of the present invention
Boundary and right margin flanking sequence.
2. the present invention analyzes and confirms the left and right border side of transgenic soybean event B5B9013-4 external source Insert Fragments for the first time
Wing sequence forms, and including No. 9 genome sequences of external source Insert Fragment sequence and the cultivated soybean Shen Nong, and determines exogenous sequences big
Specific insertion point in beans genome.
3. providing external source Insert Fragment left margin and right margin flanking sequence feature using the present invention, genetically engineered soybean is established
Event B5B9013-4 specificity qualitative PCR detection method prepares detection kit.
4. using the left and right boundary flanking sequence of external source Insert Fragment provided by the invention and method for detecting specificity, to turning
Transgenic soybean event B5B9013-4 includes parent, derivative strain or kind and its product includes plant, tissue, seed and product
Specific detection is carried out, so as to fulfill effective supervision and management to genetically engineered soybean and products thereof.
Description of the drawings
Fig. 1 .B5B9013-4 conversion carrier pTF101.1-RBSC2-P3i structure collection of illustrative plates
Fig. 2 transgenic soybean event B5B9013-4 left margin flanking sequences specific PCR detects .M：DNA molecular amount mark
Accurate (DL2000), 1：B5B9013-4 roots, 2：B5B9013-4 stems, 3：B5B9013-4 blades, 4：B5B9013-4 is spent, and 5：
B5B9013-4 seeds, 6：Soybean varieties Shen Nong 9,7：Soybean varieties Ji educates 47,8：Soybean varieties Ji educates 72,9：Maize leaf,
10：Cotton leaf
Fig. 3 transgenic soybean event B5B9013-4 right margin flanking sequences specific PCR detects .M：DNA molecular amount mark
Accurate (DL2000), 1：B5B9013-4 roots, 2：B5B9013-4 stems, 3：B5B9013-4 blades, 4：B5B9013-4 is spent, and 5：
B5B9013-4 seeds, 6：Soybean varieties Shen Nong 9,7：Soybean varieties Ji educates 47,8：Soybean varieties Ji educates 72,9：Corn, 10：
Cotton
Specific embodiment
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is further explained, but following embodiments are only the preferred embodiment of the present invention, and not all.
Based on the embodiment in embodiment, those skilled in the art obtain other realities under the premise of creative work is not made
Example is applied, belongs to protection scope of the present invention.Experimental method in following embodiments is conventional method unless otherwise specified,
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
1. transgenic soybean event B5B9013-4 exogenous sequences insertion point of embodiment is analyzed
1. genetically engineered soybean B5B9013-4 extracting genome DNAs
(1) extracting genome DNA：Take 1-2g soybean young leaflet tablets, liquid nitrogen grinding to powdered, loading 50mL centrifuge tubes
In.Sequentially add 5mL extracting solutions A (100mmol/L Tris-HCl, pH8.0,0.35mol/L sorbierites, 5mmol/L EDTA,
PH8.0,1%2- mercaptoethanol), 3.5mL extracting solutions B (50mmol/L Tris-HCl, pH8.0,4.0mol/LNaCl, 1.8%
CTAB, 25mmol/L EDTA, pH8.0), 30% sodium lauroyl sarcosines of 0.3mL and 2%PVP-360, incubate 60 at 55 DEG C~
90 minutes, during which jog was several times.Centrifuge tube is taken out, adds in isometric chloroform: isoamyl alcohol (24: 1), the jog 15 that turns upside down divides
Then clock centrifuges 10 minutes (13000rpm) under room temperature.Aspirate supernatant, add in the precooling of 2/3 volume is mixed with supernatant 1/
The isopropanol of the sodium acetate of 10 volumes, 4 DEG C of 13000rpm are centrifuged 20 minutes.Supernatant is abandoned, is rinsed with cold 75% ethyl alcohol.In air
After dry DNA to dry tack free, in -20 DEG C of preservations
(2) Genomic DNA Purification：With 200uL ddH2O dissolving DNAs add in 5uL RNase (10mg/mL), 37 DEG C of incubations
40 minutes.With isometric phenol/chloroform 1-2 times, at room temperature 13000rpm centrifugations 10 minutes.Shift supernatant to one it is new
1.5mL centrifuge tubes, and precipitated with 100% chloroform of isometric precooling.13000rpm is centrifuged 10 minutes at room temperature.Turn supernatant to new
2mL centrifuge tubes with the cold absolute ethyl alcohol of two volumes (1/10 volumes of acetic acid sodium of mixing) precipitation DNA, then place 30 in -20 DEG C
Minute.13000rpm is centrifuged 15 minutes, after 75% ethyl alcohol rinses 2 times, in air drying 15-20 minutes.50~100uL
ddH2O dissolving DNAs.After ultraviolet specrophotometer (Quawell Q5000) measures DNA concentration, saved backup in -20 DEG C.
2. genetically engineered soybean B5B9013-4 genomes weight sequencing analysis
Commission Beijing Biomarker Technologies Co., Ltd. carries out genetically engineered soybean B5B9013-4 weight sequencing analysis.It will
Qualified sample gene group DNA ultrasonic wave fragmentations are detected, then the DNA of fragmentation are purified, end is repaired, 3 ' ends
Add A, connection sequence measuring joints.Clip size selection is carried out with agarose gel electrophoresis again, PCR amplification is carried out and forms sequencing library.
The library of quality inspection qualification is sequenced using two generation high-flux sequence Xten platforms.It is original to being sequenced 40493786 obtained
Reads (both-end sequence) carries out quality evaluation, and 39143066 Clean Reads are obtained by filtration.Then by Clean Reads
With reference gene group sequence (Wm82.a2.v1, http://phytozome.jgi.doe.gov/pz/portal.html#！
infoAlias=Org_Gmax it) is compared.Positions of the Clean Reads in reference gene group, system are positioned by comparison
Count the information such as sequencing depth, the genome coverage of each sample.The Clean Data that this analysis data volume is 12.13Gbp,
Q30 reaches 85.08%.Sample and reference gene the group comparison rate that be averaged are 99.26%, and average overburden depth is 12X, and genome covers
Cover degree is 98.82% (at least one base covering).
Genetically engineered soybean B5B9013-4 weight sequencing datas are compared into reference gene group and external source insetion sequence, root respectively
Following two classes Paired_end reads are found out according to comparison result：The first kind is reference gene group sequence in the reads comparisons of one end,
Insetion sequence in other end reads comparisons；Second class is reference gene on any a part of sequence alignments of one end reads in both ends
Group sequence, another part compare upper insetion sequence.Reference gene group is compared using bwa, selection can compare external source insetion sequence
Whole reads, carry out local assembling.External source insetion sequence and reference are compared using blastn according to the contig of assembling respectively
Genome is as a result, choose contig sequence alignments to the region of chromosome, and carry out IGV to the bwa comparison results in these regions
Sectional drawing is verified, obtains external source Insert Fragment insertion position information.Analysis result shows genetically engineered soybean B5B9013-4 external source pieces
Section insertion position is 46747946 site of Chr04 chromosomes, and inserted mode is inserted into for double copy unit points, upstream and downstream~
2kb sequences (Gm04：46748946..46746945) as shown in SEQ-1.
The 2. left and right boundary flank sequence analysis of transgenic soybean event B5B9013-4 external source Insert Fragments of embodiment
According to transgenic soybean event B5B9013-4 external sources insetion sequence and insertion point in soybean reference gene group
Upstream and downstream sequence designs PCR detection primers.B5B9013-4 insertion point upstream sequences amplimer is B5B9013LB-F1
(5 '-CCCTCACTCCATTTGTCCTCT-3 ') and B5B9013LB-R1 (5 '-TCCCACATACTTCCTCCCTCT-3 ')；
B5B9013-4 insertion point downstream sequences amplimer is B5B9013RB-F1 (5 '-TACTTCCTCCCTCTTCAGCACC-3 ')
With B5B9013RB-R1 (5 '-GGTAGCGATGTGGACCTTAGCCTT-3 ').
Using B5B9013-4 genomic DNAs template, PCR amplification is carried out respectively using above-mentioned primer.PCR reaction systems
(25uL) is：10 × PCR buffer solutions 2.5uL, 10mmol/L dNTPs 0.5uL, 5U/uL Taq enzyme 0.5uL, sample DNA
1.0uL, 10umol/L forward primer 0.5uL, 10umol/L reverse primer 0.5uL, ddH2O 19.5uL.PCR reaction conditions are：
95℃ 5min；94 DEG C of 45s, 60 DEG C of 45s, 72 DEG C of 3min, totally 35 recycle；72℃ 15min.Utilize 1% Ago-Gel
Electrophoresis detection pcr amplification product.Then using plastic recovery kit purified pcr product, and it is connected to the EZ-T of GENSTAR companies
Cloning vector.Shanghai life work is entrusted to carry out sequence verification, and by sequencing result and external source insetion sequence and reference gene group sequence
It compares, the final transgenic soybean event B5B9013-4 external source Insert Fragment left margin flanking sequences that obtain are right as shown in SEQ-2
Boundary flanking sequence is as shown in SEQ-3.The sequence origin is derived from soybean genomic sequence and from external source Insert Fragment sequence
Arrange the DNA sequence dna of composition.
B5B9013-4 external sources Insert Fragment left margin flanking sequence (SEQ-2) length is 1041bp, wherein, 1-1071
Site sequence derives from No. 9 genome sequences of the cultivated soybean Shen Nong, and 1072-1401 site sequences derive from external source Insert Fragment
Sequence.B5B9013-4 external sources Insert Fragment right margin flanking sequence (SEQ-3) length is 845bp, wherein, 1-323 sites sequence
Row derive from No. 9 genome sequences of the cultivated soybean Shen Nong from external source Insert Fragment sequence, 324-845 site sequences.
3. transgenic soybean event B5B9013-4 specific PCRs of embodiment detect
According to transgenic soybean event B5B9013-4 external source Insert Fragment left margin flanking sequences (as shown in SEQ-2) and
Right margin flanking sequence (as shown in SEQ-3), separately designs specific detection primer.Left margin flanking sequence specific detection is drawn
In object combination, wherein one article of primer is the forward primer according to the design of SEQ-2 1-1071 site sequences, as shown in SEQ-4；Separately
One article of primer is the reverse primer according to the design of SEQ-2 1072-1401 site sequences, as shown in SEQ-5.Right margin flank sequence
In the combination of row specific detection primer, wherein one article of primer is the forward primer according to the design of SEQ-3 1-323 site sequences,
As shown in SEQ-6；Another article of primer is the reverse primer according to the design of SEQ-3 324-845 site sequences, as shown in SEQ-7.
Extraction Transgenic soybean plants B5B9013-4 roots, stem, leaf, flower and seed DNA sample respectively, DNA extraction method according to
Method in embodiment 1.47, Ji is educated with receptor Non-transgenic soybean kind Shen Nong 9, conventional soy kind Ji and educates 72 and corn
With cotton as compareing, PCR amplification is carried out.PCR reaction systems (25uL) are：10 × PCR buffer solutions 2.5uL, 10mmol/L
DNTPs 0.5uL, 5U/uL Taq enzyme 0.5uL, DNA sample 1.0uL, 10umol/L forward primer 0.5uL, 10umol/L is reversed
Primer 0.5uL, ddH2O19.5uL.PCR reaction conditions are：95℃ 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 45s, totally 30
A cycle；72℃ 5min.Whether PCR product is detached through 1% agarose gel electrophoresis, and is dyed using EB, deposited with identification
In specific amplification band.When carrying out PCR amplification with above-mentioned specific primer, Non-transgenic soybean kind Shen Nong 9, routine are big
Beans kind Ji educate 47, Ji educate 72 and corn and cotton without amplified band, only genetically engineered soybean B5B9013-4 samples include
Root, stem, leaf, flower and seed generate specific amplification band.Wherein, left margin flanking sequence expanding fragment length is 355bp, such as
Shown in Fig. 2；Right margin flanking sequence expanding fragment length is 316bp, as shown in Figure 3.The study show that it is inserted into piece using external source
Whether section flanking sequence specific primer carries out PCR analyses, can specifically detect in sample and contain from B5B9013-4
Ingredient.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
God and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (8)
1. wide spectrum mosaic disease resisting poison transgenic soybean event B5B9013-4 external source Insert Fragment left margins and right margin flanking sequence
As shown in SEQ-2 and SEQ-3, which is characterized in that origin is derived from soybean genomic sequence and from external source Insert Fragment sequence
The DNA sequence dna of composition；Wherein：
External source Insert Fragment left margin flanking sequence feature includes：
(1) SEQ-2 1-1071 site sequences derive from No. 9 genome sequences of the cultivated soybean Shen Nong；
(2) SEQ-2 1072-1401 site sequences derive from external source Insert Fragment sequence；
External source Insert Fragment right margin flanking sequence feature includes：
(1) SEQ-3 1-323 site sequences derive from external source Insert Fragment sequence；
(2) SEQ-3 324-845 site sequences derive from No. 9 genome sequences of the cultivated soybean Shen Nong.
2. the preparation method of the left and right boundary flanking sequence of external source Insert Fragment described in claim 1, which is characterized in that extraction turns
Transgenic soybean event B5B9013-4 genomic DNAs determine exogenous sequences insertion point using genome weight sequencing technologies analysis,
And it is obtained by PCR amplification.
3. sequence shown in claims 1 is establishing transgenic soybean event B5B9013-4 method for detecting specificity or is preparing inspection
Application in test agent box, which is characterized in that according to sequence shown in claims 1, prepare specific primer or probe.
4. sequence described in claim 1, method for detecting specificity described in claims 3 or detection kit are in genetically engineered soybean
Event B5B9013-4 includes parent, derivative strain or kind and its product is included in plant, tissue, seed and product testing
Using.
5. method for detecting specificity or detection kit described in claim 3, claim 4, which is characterized in that wherein one is drawn
Object is the forward primer according to the design of SEQ-2 1-1071 site sequences, and another article of primer is according to SEQ-2 1072-1401
The reverse primer of site sequence design, i.e., described two primers are combined as external source Insert Fragment left margin described in claims 1
Flanking sequence specific detection primer.
6. external source Insert Fragment left margin flanking sequence specific detection primer according to claim 5, it is characterised in that：Such as
Shown in SEQ-4 and SEQ-5：
The forward primer SEQ-4 is：5’-ACGCAAGCAGATACTTCACCT-3’
The reverse primer SEQ-5 is：5’-TCAGATTGTCGTTTCCCGCC-3’.
7. method for detecting specificity or detection kit described in claim 3, claim 4, which is characterized in that wherein one is drawn
Object is the forward primer according to the design of SEQ-3 1-323 site sequences, and another article of primer is according to SEQ-3 324-845 sites
The reverse primer of sequence design, i.e., described two primers are combined as external source Insert Fragment right margin flank described in claims 1
Sequence-specific detection primer.
8. external source Insert Fragment right margin flanking sequence specific detection primer according to claim 7, which is characterized in that such as
Shown in SEQ-6 and SEQ-7：
The forward primer SEQ-6 is：5’-TCCACACAACATACGAGCCG-3’
The reverse primer SEQ-7 is：5’-GCATACATCACATTCACACCCA-3’.
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