CN100529089C - Flanking sequence of exogenous event inserting vector for transgenic rape Topas-19/2 and its application - Google Patents
Flanking sequence of exogenous event inserting vector for transgenic rape Topas-19/2 and its application Download PDFInfo
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Abstract
The present invention discloses flanking sequence of exogenous event inserting vector for transgenic rape Topas 19/2 and its application, and relates to bioengineering technology. The present invention obtains flanking sequence, SEQ No. 1, of exogenous event inserting vector for transgenic rape Topas 19/2 with transgenic rape Oxy-235 as material, GenomeWalker of Invitrogen co. for constituting genomic library, and primer pair TOPLB-1 and TOPLB-2, and the jointing primer, and through PCR amplification. The present invention designs primers TOPLG and TOPLV and TaqMan probe TOPLP; and establishes the specific quantitative and qualitative detection method of exogenous event for transgenic rape Topas 19/2. The present invention is suitable for specific PCR detection of other Topas 19/2 events.
Description
Technical field
The present invention relates to the detection of transgene rape in the technical field of bioengineering, specifically, relate to exogenous insertion vector flanking sequence and the application thereof of a kind of transgene rape exogenous origin gene integrator incident Topas 19/2.
Background technology
In recent years, genetically modified crops such as corn, soybean, cotton, rape and tomato are got permission plantation and production in a lot of countries, and what have is processed to food, feed or is used as foodstuff additive.Because the ecological safety and the edible safety of transgenic product are controversial always, more than 30 countries and regions are implemented transgenic product sign system in succession.
According to the sequence information of foreign gene in the transgenic product, the array mode of foreign gene on transgenic constructs, and the integration site of construct on genome can adopt the detection method of gene specific, carrier specificity and event-specific.
1, gene specific detection method
Because a large amount of transgene carriers used identical foreign gene, so gene specific detection method and be not suitable for the reality that current transgenic product continues to bring out.
2, carrier specificity detection method
The carrier specificity detection method is utilized the border sequence feature between the gene element, can effectively identify the different constructs that use similar gene element, and the characteristics that exist sequence information to obtain easily, is therefore used by Many researchers.But, may obtain the transformant of a more than different qualities by same construct, even may be transformed in the different species, and these transformants may not all pass through safety assessment.Therefore the carrier specificity detection method can not be discerned different transgenic strains, can not differentiate this transgenic strain and whether pass through safety assessment.
3, the detection method of event-specific
The basis that event-specific detects is that the integration site of transgenic constructs on genomic dna sequence has high degree of specificity, even identical carrier repeatedly transforms, the position of integration and integration site feature also are unrepeatable.Therefore, according to unrepeatable integration site characteristic sequence, the specific detection method of the incident of having developed.Compare with preceding two kinds of methods, event-specific detects has the highest specificity, the most suitable qualitative and detection by quantitative of doing transgenic product.But the complete information of transgenic constructs is difficult to obtain usually, and known sequence gene element may have suitable distance apart from the border, and in the process of integrating, complicated reorganization may take place between carrier and the genome, therefore, separate flanking sequence and become the key that event-specific detects.In February, 2000, European Union has subsidized Qpcrgmofood European Project project for this reason, and this project is now successfully separated a plurality of kind flanking sequences of acquisition and is used to set up the event-specific detection method.Current, the event-specific detection method has been used to transgenosis Roundup Ready soybean, the detection of a series of transgenosis commercialization kinds such as Mon810 maize.
Through retrieval, do not find any about transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector with utilize this sequence to set up the report that event-specific is qualitative, quantitative PCR (polymerase chain reaction) detects as yet to existing patent and other documents.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists in detecting transgene rape Topas 19/2 incident and transgene rape kind Topas 19/2, a kind of transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector and application thereof are provided.
The object of the present invention is achieved like this:
The present invention is a material with transgene rape kind Topas 19/2, utilize the GenomeWalker of Invitrogen company test kit to make up genomic library, and utilize primer: TOPLB-1:5 ' TCGCCCAATAGCAGCCAGTCCCTTCC 3 ' and TOPLB-2:5 ' AGTCCCTTCCCGCTTCAGTGACAACG 3 ' and the joint primer that test kit provides, obtain Topas 19/2 flanking sequence of exogenous event inserting vector SEQ NO.1 with round pcr (polymerase chain reaction) amplification.
The feature of transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector is:
(1) the 1st to 550 bases derive from the exogenous insertion vector sequence;
(2) the 551st to 823 bases derive from the rape genome sequence;
(3) origin the 551st to 823 base coming from the 1st to 550 base of exogenous insertion vector and derive from the rape genome sequence formed rape Topas 19/2 flanking sequence of exogenous event inserting vector jointly.
The applied research of above-mentioned transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector:
(1) utilize this sequence signature to design the event-specific qualitative PCR detection method of transgene rape exogenous origin gene integrator incident Topas 19/2;
(2) utilize this sequence signature to design the event-specific quantitative PCR detecting method of transgene rape exogenous origin gene integrator incident Topas 19/2;
(3) utilize this sequence signature to design the strain specificity qualitative PCR detection method of transgene rape kind Topas 19/2;
(4) utilize this sequence signature to design the strain specificity quantitative PCR detecting method of transgene rape kind Topas 19/2.
Compared with prior art, the advantage and the effect that have of the present invention is as follows:
1, the present invention checks order first and announces transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector.
2, the present invention analyzes and confirms the source of different bases in transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector first, and the joint site of definite exogenous insertion vector sequence and rape genome sequence.
3, the sequence signature that utilizes the present invention to find is set up qualitative, the quantitative PCR detecting method of event-specific of transgene rape Topas 19/2 incident and transgene rape kind Topas 19/2.
4, the present invention is applicable to the aspects such as other event-specifics PCR detection method of setting up transgene rape Topas 19/2 incident and transgene rape kind Topas19/2.
Description of drawings
Fig. 1-transgene rape Topas 19/2 incident binding site, its binding site characteristic sequence.
Fig. 2-transgene rape Topas 19/2 event-specific qualitative PCR amplification,
Wherein:
M-molecular weight Marker;
1-transgenic rape Ms 8 Rf3 genomic dna template;
2-transgenic rape Ms 1 Rf1 genomic dna template;
3-transgenic rape Ms 1 Rf2 genomic dna template;
4-transgene rape Topas 19/2 genomic dna template;
5-transgene rape T45 genomic dna template;
6-transgene rape Topas 19/2 genomic dna template;
7-transgene rape RT45 genomic dna template:
Oily 821 templates in the 8-non-transgenic rape.
Fig. 3-transgene rape Topas 19/2 event-specific quantitative pcr amplification.
Embodiment
1, the pcr amplification of transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector
Earlier 20%SDS is preheating to 65 ℃, gets 15ml SDS extracting buffer (0.1TrisHCl, 0.05MEDTA, 1M NaCl pH8.0) and join the 50ml centrifuge tube, add 2.5 μ l beta-mercaptoethanols again, mixing; Blade about liquid nitrogen grinding 3g goes to 50ml with powder and contains in the 50ml centrifuge tube of extracting buffer, and the mixing that vibrates on vibrator adds the 20%SDS of 2ml preheating, mixing, and 65 ℃ of water-baths at least 30 minutes will be shaken test tube therebetween gently; After the water-bath, rapidly centrifuge tube is placed on ice, add 3ml 3M KAc, mixing was placed 30 minutes on ice; 4 ℃ of centrifugal 5min of 5000g; Supernatant is transferred in the new 50ml centrifuge tube, added the Virahol of 2/3 volume, mixing is placed more than the 30min for-20 ℃; 6000g, 4 ℃ of centrifugal 15min outwell supernatant, wash one time with 75% ethanol, and the ultrapure water dissolving DNA is used in vacuum-drying, after the dissolving, solution is transferred in the centrifuge tube of 15ml; Add Proteinase K by 1% of DNA volume of dissolution, 55 ℃ of water-bath 30min; Add equal-volume phenol, mixing, jog 30min, the centrifugal 10min of 8000g; Shift supernatant to new tube, add isopyknic phenol-chloroform-primary isoamyl alcohol (25: 24: 1), jog 20min, the centrifugal 15min of 8000g; Shift supernatant to new tube, add isopyknic chloroform-primary isoamyl alcohol (24: 1), jog 20min, the centrifugal 15min of 8000g; Draw supernatant, every pipe adds 5 μ l RNase, mixing, 37 ℃ of water-bath 1hr degradation of rna; With isopyknic phenol extracting once, jog 20min, the centrifugal 15min of 8000g; Shift supernatant to new centrifuge tube, add isopyknic chloroform-primary isoamyl alcohol (24: 1), jog 20min, the centrifugal 15min of 8000g; Suct and reset and add into 1/10 volume 3M NaAC, mixing adds the equal-volume Virahol, places 30min, deposit D NA for-20 ℃; 6000g, 4 ℃ of centrifugal 15min outwell supernatant, wash 2 times with 75% ethanol, centrifugally remove 75% ethanol, vacuum-drying; After the drying, standby with the ultrapure water dissolving DNA.
Utilizing the GenomeWalker of Invitogen company test kit, is material with rape Topas 19/2 genomic dna, and the method that provides according to test kit makes up rape Topas 19/2 genomic library.
The synthetic primer sequence is as follows: TOPLB-1:5 ' TCGCCCAATAGCAGCCAGTCCCTTCC 3 ' and TOPLB-2:5 ' AGTCCCTTCCCGCTTCAGTGACAACG 3 '.
With Topas 19/2 genomic library is template, and the joint primer AP1 and the TOPLB-1 that utilize test kit to provide carry out pcr amplification.In the 50ul reaction system, get Topas 19/2 genomic library DNA 1ul, other each component final concentrations are KOD Plus Buffer 1x, every kind of 200uM of dNTPs, MgSO
41mM, each 100nM of primer, KOD Plus enzyme 1u.Reaction conditions is, 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 15 seconds, 68 ℃ 3 minutes, circulate 68 ℃ of insulations 7 minutes 45 times.Get 1ul PCR product, 50 times of dilute with waters are therefrom got 1ul as second template of taking turns PCR.Second to take turns reaction be primer with AP2 and the TOPLB-2 that test kit provides, and reaction system is identical with the first round.Reaction conditions is, 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 15 seconds, 68 ℃ 3 minutes, circulate 68 ℃ of insulations 7 minutes 25 times.The agarose electrophoresis DNA isolation, the EB evaluation of dyeing.
With the QIAquick PCR Purification Kit of QIAGEN company purified pcr product.
PZero2 (Invitrogen) carrier that PCR product and EcoRV enzyme are cut is connected.Linked system: DNA mixture 22 μ l; 2.5 μ l 10 * T4 DNA Ligase Buffer with 1mM ATP; 0.5 μ lT4 DNA Ligase (NEB).22 ℃ connect 1hr at least.
The method that provides according to " molecular cloning experiment guide " prepares intestinal bacteria TOP10 (Invitrogen) competent cell, and transforms to connect product.The picking mono-clonal carries out bacterium colony PCR with the mutational site special primer and detects recon.With the clone's enlarged culturing that filters out, prepare plasmid in a small amount, enzyme is cut checking.
Send the order-checking of order-checking company with the clone who filters out.With the sequence information that obtains, the Blastn software that utilizes NCBI to provide is analyzed the source of different piece sequence, thereby judges the binding site of genome and carrier.
2, application method
1) utilizes the event-specific qualitative PCR detection method that sequences Design transgene rape Topas 19/2 incident and transgene rape kind Topas 19/2 are provided among the present invention
The synthetic primer sequence is as follows: TOPLG:5 ' CGGCCTTAATCCCACCCCAG 3 ' and TOPLV:5 ' AGTTCCAAACGTAAAACGGCTT 3 '.
Get transgene rape kind Ms8Rf3 respectively, Ms1Rf1, Ms1Rf2, Topas 19/2, T45, Topas19/2, GT73; Oil 821 in the non-transgenic rape variety, and Arabidopis thaliana, Chinese cabbage, wild cabbage, leaf mustard, soybean, paddy rice, corn, cotton genomic dna are template, utilize the TOPLG/TOPLV combination of primers to carry out pcr amplification respectively.In the 25ul reaction system, be template with 100ng different sources genomic dna, other each component final concentrations are PCR Buffer 1x (containing 10mM TrisHCl pH8.3, KCl 50mM), every kind of 200uM of dNTPs, MgCl
22.5mM, each 250nM of primer, Hot Start Taq enzyme 1u.Reaction conditions is, 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 15 seconds, 60 ℃ 30 seconds, 72 ℃ 30 seconds, circulate 72 ℃ of insulations 2 minutes 35 times.The PCR product separates with agarose gel electrophoresis, and EB dyeing back identifies whether there is amplified production.
2) utilize the event-specific quantitative PCR detecting method that sequences Design transgene rape Topas 19/2 incident and transgene rape kind Topas 19/2 are provided among the present invention
Synthetic TaqMan probe sequence is as follows: TOPLP:5 ' FAM-TCCCGGTCATATATCAGCGCCGGTC-TAMRA 3 '.
Primer, probe combinations at Topas 19/2 flanking sequence of exogenous event inserting vector are used to the quantitative fluorescent PCR analysis.The quantitative fluorescent PCR analysis is carried out on MJR DNA Engine Opticon 2 ContinuousFluorescence Detector, and detection and analysis software are Opticon Monitor 2 Version2.02.
Topas 19/2 incident detection by quantitative reaction volume 20ul contains template DNA 100ng, and other component concentrations are: TaqMan Buffer 1x (50mM KCl, 10mM.Tris-HCl, 10mM EDTA, pH 8.3), MgCl
24mM, primer TOPLG and TOPLV 100uM, probe TOPLP 50uM, each 200uM of dATP dCTP dGTP, dUTP 400uM, Amperase Uracil N-glycosylase (UNG) 0.2u, AmpliTaqGold 1.25u.
The TaqMan reaction conditions is: after 50 ℃ of 2 minutes and 95 ℃ of pre-sex change in 10 minutes, carry out 50 PCR circulations: 95 ℃ of sex change in 15 seconds, plate is read in 60 ℃ of annealing in 1 minute and extending.
Transgene rape Topas 19/2 genomic dna is diluted to different content by same concentrations non-transgenic rape 821 DNA, is template with the mixed rape genomic dna of 100ng, carries out the quantitative fluorescent PCR reaction.Different extension rates contain 100,13,1.3,0.13,0.013 respectively, and the mixed DNA sample of 0.0065ng Topas 19/2 genomic dna is used to set up typical curve.All fluorescent quantitation reactions all repeat 3 times.
Result according to typical curve is optimized utilizes the PCR reaction conditions identical with setting up typical curve, is with reference to the amount of measuring Topas 19/2 genomic dna in the mixed rape DNA sample that contains Topas 19/2 genomic dna with the typical curve.Getting the 100ng genomic dna is template, utilizes the different content standard model to do typical curve, according to the typical curve that obtains and the Ct value of transgenosis sample, calculates the quality of Topas 19/2 genomic dna, thereby calculates the content of Topas 19/2 in sample.All fluorescent quantitation reactions all repeat 3 times.
3. experimental result
1) pcr amplification and the sequencing analysis of transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector
Utilize AP1/TOPLB-1 and AP2/TOPLB-2 combination of primers, the GenomeWalker library that makes up with Topas 19/2 genomic dna is a template, and successfully amplification obtains the 823bp amplified production.To PCR product order-checking, and analyze through Blastn, find wherein 550 base pairs and different sources carrier sequence homology, its left margin tumor-necrosis factor glycoproteins is lost, and all the other 273 base pairs and Chinese cabbage genome sequence homology are considered to the rape genome sequence.Concrete transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector is seen SEQ NO.1.
According to above analysis, the sequence that the present invention separates acquisition comprises transgene rape Topas 19/2 exogenous event inserting vector binding site, and its binding site characteristic sequence as shown in Figure 1.
2) utilize the event-specific qualitative PCR detection method that sequences Design transgene rape Topas 19/2 incident and transgene rape kind Topas 19/2 are provided among the present invention
With different sources transgene rape, non-transgenic rape genomic dna is template, utilizes Topas 19/2 flanking sequence of exogenous event inserting vector Auele Specific Primer combination TOPLG/TOPLV, carries out pcr amplification, result such as Fig. 2.Have only Topas 19/2 genomic dna can amplify special PCR product, and other transgenosiss and non-transgenic rape DNA all there are not amplified production can be observed when doing template.Be template with other non-rape plant genome DNAs simultaneously, also do not have the visible pcr amplification product.Therefore, we think that this combination of primers has good specificity, are suitable for the detection of event-specific.
3) utilize the event-specific quantitative PCR detecting method that sequences Design transgene rape Topas 19/2 incident and transgene rape kind Topas 19/2 are provided among the present invention
According to the gradient dilution template, the fluorescence curve information that repeats to obtain for 3 times is established at the specificity fluorescent quantitative PCR reaction normal curve of Topas 19/2 incident, as shown in Figure 3, and its R
2Value is 0.995, shows that the copy number of foreign gene and fluorescence intensity all have good corresponding relation, are suitable for the accurate quantification of foreign gene.
In order to verify the accuracy of the quantivative approach of setting up in this research, we have used the genomic dna that extracts from standard transgene rape Topas 19/2 product to be diluted to certain content with non-transgenic rape genomic dna, and do contrast with the same amount genomic dna that does not contain transgene rape Topas 19/2 genomic dna, template as the real-time fluorescence quantitative PCR reaction, and, calculate the content of transgene rape Topas 19/2 genomic dna in the different samples according to the typical curve that the same terms obtains down.
With the non-transgenic rape genomic dna that do not contain transgene rape Topas 19/2 is template, does not have amplified production to be detected.For mixed laboratory sample, detect with setting up Topas 19/2 event-specific fluorescence quantitative detecting method in this research, all very approaching with theoretical value, error is less than 15%.
Above result as can be seen, the present invention has been for the quantitative detecting analysis of transgene rape incident Topas 19/2 and transgene rape Topas 19/2 provides based on simple, measuring method reliably, can be used for mixed product transgene rape Topas 19/2 incident of different sources, different content and transgene rape kind Topas 19/2 quantitatively.The present invention provides a kind of useful reference for transgenosis sign, provides necessary means to the control of transgenic product.
Sequence table
＜110〉Inst. of Oil Crops, Chinese Academy of Agriculture
＜120〉transgene rape Topas 19/2 flanking sequence of exogenous event inserting vector and application thereof
<160>1
<210>1
<211>823
<212>DNA
＜213〉rape (Brassica napus cv.Topas 19/2)
<400>
gtcccttccc?gcttcagtga?caacgtcgag?cacagctgcg?caaggaacgc?ccgtcgtggc 60
cagccacgat?agccgcgctg?cctcgtcctg?cagttcattc?agggcaccgg?acaggtcggt 120
cttgacaaaa?agaaccgggc?gcccctgcgc?tgacagccgg?aacacggcgg?catcagagca 180
gccgattgtc?tgttgtgccc?agtcatagcc?gaatagcctc?tccacccaag?cggccggaga 240
acctgcgtgc?aatccatctt?gttcaatcca?catgatcaga?tctggattga?gagtgaatat 300
gagactctaa?ttggataccg?aggggaattt?atggaacgtc?agtggagcat?ttttgacaag 360
aaatatttgc?tagctgatag?tgaccttagg?cgacttttga?acgcgcaata?atggtttctg 420
acgtatgtgc?ttagctcatt?aaactccaga?aacccgcggc?tcagtggctc?cttcaacgtt 480
gcggttctgt?cagttccaaa?cgtaaaacgg?cttgtcccgc?gtcatcggcg?ggggtcgtaa 540
cgtgactccc?ggtcatatat?cagcgccggt?cggccccggg?cctggggtgg?gattaaggcc 600
gaaggcccga?acccatccta?gtctacaaaa?aaaaaaaaaa?aaaaaaacta?accacgtaaa 660
gaccaacaga?tttaccgttg?cagattaaac?gaccataaga?ggagtagttt?ttccttgtta 720
agaatagtga?acatggcatg?tcatcaaagt?tacacgtggg?aatatacgac?atttacacgt 780
tgacttaatg?gaacacagct?gttgagtttt?tgtccagtag?ttt 823
Claims (3)
1, a kind of transgene rape Topas 19/2 exogenous event inserting vector side dna fragmentation is characterized in that:
Base sequence is shown in SEQ NO.1, and origin comes from the 1st to 550 base of exogenous insertion vector sequence and forms jointly with the 551st to 823 base that derives from the rape genome sequence.
2,, it is characterized in that utilizing the event-specific qualitative PCR detection method of the transgene rape exogenous origin gene integrator incident Topas 19/2 of this sequence signature design by the application of the described transgene rape Topas 19/2 exogenous event inserting vector side dna fragmentation of claim 1:
The synthetic primer sequence is as follows: TOPLG:5 ' CGGCCTTAATCCCACCCCAG 3 ' and TOPLV:5 ' AGTTCCAAACGTAAAACGGCTT 3 '; Extract sample total DNA, utilize the TOPLG/TOPLV combination of primers to carry out pcr amplification respectively; The PCR product separates with agarose gel electrophoresis, and EB dyeing back identifies whether there is amplified production, as exists amplified production then to contain the composition of transgene rape Topas 19/2 Event origin in the interpret sample.
3,, it is characterized in that utilizing the event-specific quantitative PCR detecting method of the transgene rape exogenous origin gene integrator incident Topas 19/2 of this sequence signature design by the application of the described transgene rape Topas 19/2 exogenous event inserting vector side dna fragmentation of claim 1:
The synthetic primer sequence is as follows: TOPLG:5 ' CGGCCTTAATCCCACCCCAG 3 ' and TOPLV:5 ' AGTTCCAAACGTAAAACGGCTT 3 '; Synthetic TaqMan probe sequence is as follows: TOPLP:5 ' FAM-TCCCGGTCATATATCAGCGCCGGTC-TAMRA 3 '; Extract sample total DNA, utilize above-mentioned primer TOPLG/TOPLV and probe TOPLP combination to carry out fluorescent quantitative PCR respectively; Transgene rape Topas 19/2 genomic dna is diluted to different content by same concentrations non-transgenic rape 821DNA, is template with the mixed rape genomic dna of 100ng, carries out the quantitative fluorescent PCR reaction and sets up typical curve; With the typical curve is with reference to the amount of measuring Topas 19/2 genomic dna in the mixed rape DNA sample that contains Topas 19/2 genomic dna.
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| CN109825631A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgene rape strain Topas19-2 detection method and reagent |
| CN110527737B (en) * | 2019-08-21 | 2024-03-08 | 中国农业科学院油料作物研究所 | Positive plasmid molecule pYCID-1905 identified by transgenic rape and transformant of transgenic rape product and application thereof |
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