CN102409081B - A kind of be used for genetically engineered soybean and transgene cotton detection standard plasmid molecule and construction process thereof - Google Patents
A kind of be used for genetically engineered soybean and transgene cotton detection standard plasmid molecule and construction process thereof Download PDFInfo
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Abstract
The invention discloses a kind of for genetically engineered soybean and cotton detection standard plasmid molecule and construction process thereof.Described standard plasmid molecule contains eight gene-specific fragment (Lec1,35S, NOS, PAT, Sad1, Cry1Ab/c and EPSPS) of soybean and cotton.By fusion DNA vaccine method, these eight gene fusion are become a large fragment, be cloned on plasmid vector and obtain recombinant plasmid molecule pTLE8.The standard plasmid molecule built in the present invention can replace positive criteria product to be applicable to screening to transgenic soybean lines GTS40-3-2 and Bt gene cotton, the quantitative and qualitative analysis pcr analysis of gene specific and strain specificity and detection.Occur if any new transgenic line, new exogenous genetic fragment can be added on the basis of primary standard molecule, meet the detection needs of increasing genetically modified crops material.
Description
Technical field
The invention belongs to technical field of molecular biology, specifically, relate to a kind of for genetically engineered soybean and transgene cotton detection standard plasmid molecule and construction process thereof.
Background technology
Since the first transgenic Fructus Lycopersici esculenti " FLAVR SAVR " comes out, along with the development of biotechnology, genetically modified crops (Genetically Modified Plants, GMP) are more and more applied to agriculture field.From commercialization first in 1996 successfully by 2009, the product of antiweed is occupied an leading position genetically modified crops always, is secondly anti insect gene.According to statistics, the genetically modified crops of whole world approval plantation in 2009 are about 1.34 hundred million hectares, and wherein genetically engineered soybean cultivated area reaches 6,900 ten thousand hectares, accounts for 77% of the total cultivated area of soybean.Transgene cotton cultivated area is 1,617 ten thousand hectares, accounts for 49% of the cotton total area; The transgene cotton of China's plantation in 2009 is mainly Insect Resistant Cotton, accounts for 68% of total cultivated area.Along with the extensive plantation of genetically modified crops, its safety issue causes the most attention of the global public, international organization and country formulate corresponding security control regulation one after another, strengthen the standardized administration of genetically modified crops, and implement mark system to transgenic plant and products thereof.China has issued " agriculture GMO bio-safety management rules " May 23 calendar year 2001, go into effect on March 20th, 2002 " agriculture genetically modified organism identity management way ", " agriculture genetically modified organism import security management process " and " agriculture GMO bio-safety evaluation management method ", requiring tests to 5 large classes, 17 kinds of genetically modified crops and products thereof quarantines and mark, and the detection method therefore setting up various product transgene component is particularly urgent.At present, based on the PCR detection technique of nucleic acid because having the advantage of highly sensitive and high specificity, most important, most widely used genetically modified crops and products thereof detection technique at present has been become.
For the exogenous DNA array inserted, PCR inspection policies can be divided into four kinds, and namely universal component selective mechanisms, gene specific detect, build specific detection and event-specific detection.Universal component screening PCR detects mainly gene fragment for the purpose of transgenosis universal component CaMV35S promotor and NOS terminator; It is that the DNA fragment specific inserting foreign gene detects fragment as object that gene specific PCR detects; It is realized by the joining region sequence detecting exogenous insertion vector and Plant Genome that strain specificity PCR detects.Due to each Transgenic Plant Lines, all there is the joining region sequence of special exogenous insertion vector and Plant Genome, and joining region sequence is single copy, so event-specific detection method has higher specificity and accuracy.Event-specific detection has become the emphasis of current detection GMOs research, and step by step by international each testing laboratory is adopted.
When carrying out PCR and detecting, need to arrange positive control with the reliability of the validity and detected result of guaranteeing testing process, particularly need in quantitative PCR detection with content accurately genetically modified crops positive criteria product (CRMs) carry out production standard curve.But because genetically modified crops exist biological safety and the restriction with prior art, genetically modified crops positive criteria product are difficult to obtain, and this present situation has become the bottleneck of detection method and application.Therefore the novel positive control material that development can substitute genetically modified crops reference material is needed badly, to meet the detection GMOs demand of development.
At present, gain public acceptance in International Plant GMO detection using plasmid as positive control.In the article that International Periodicals is delivered, a lot of positive control employs standard plasmid molecule.The Objective Concept Japanese Scientists Kuribara of standard molecule equals to propose for 2002, it refers to a kind of linearizing recombinant plasmid molecule containing the specific fragment of detection GMOs object foreign gene and/or internal standard gene, and research shows that plasmid control molecule is good reference material surrogate in GMO identification and detection.Relative to traditional positive criteria material, plasmid control molecule is used for transgene component quantitative analysis and has following advantage: first, and preparation process not by the restriction that transgenic plant growth cycle and plantation are permitted, and can steady in a long-termly be preserved; Secondly, purity and the amount of the plasmid DNA extracted from engineering bacteria more easily control compared with the gDNA extracted from vegetable material and more easily preserve, decrease the step extracting gDNA from positive criteria material in use, thus save experimental period and avoid gDNA and extract the experimental error caused.Again, due to the molecular weight of plasmid, during use, the gDNA of concentration ratio identical copies number is low, and experiment requirement is considerably less, greatly can reduce the cost of experiment.The standard plasmid molecule built can relate to multiple genes of the genetically modified crops of different plant species simultaneously.Utilize fusion DNA vaccine technology can realize the splicing of multiple gene fragment, utilize the detection that a standard plasmid molecule just can realize multiple genetically modified crops like this.
Summary of the invention
The object of the present invention is to provide a kind of genetically engineered soybean, transgenic Bt cotton detection standard plasmid molecule and construction process thereof, make it that positive criteria material can be replaced to detect for the quantitative and qualitative analysis PCR of genetically engineered soybean, transgenic Bt cotton.Utilize the PCR of the principle design of fusion DNA vaccine technology soybean Lectin gene, 35S promoter, NOS terminator, pat gene, EPSPS gene, transgenic soybean lines GTS40-3-2, cotton Sad gene and Cry1Ab/c fusion gene to merge primer, obtain the fusion large fragment of eight genes through fusion DNA vaccine amplification.Utilize molecule clone technology that fusion large fragment is inserted into pMD
tMin 19-T Simple carrier, obtain recombinant plasmid molecule pTLE8.The standard plasmid molecule pTLE8 that the present invention builds can as the positive control in testing process, and the difficult problem detecting Plays material want for solving genetically modified crops provides an effective way and effectively can ensure the carrying out of testing.
The present invention is realized by following experimental program, standard plasmid molecule of the present invention, with alternative transgenic soybean lines GTS40-3-2, the reference material turning PAT soybean and transgenic Bt cotton, detects for quantitative and qualitative analysis PCR.The one section sequence of this standard plasmid molecule simultaneously containing soybean reference gene Lectin, 35S promoter, NOS terminator, pat gene, EPSPS gene, transgenic soybean lines GTS40-3-2, cotton internal standard gene Sad and Cry1Ab/c fusion gene.
A kind of genetically engineered soybean of the present invention, transgenic Bt cotton detection standard plasmid molecule and construction process thereof, comprise the following steps:
(1) utilize GenBank database lookup and obtain the sequence of soybean reference gene Lectin, the strain specificity sequence of transgenic soybean lines GTS-40-3-2,35S promoter, NOS terminator, EPSPS gene, pat gene, cotton reference gene Sad and Cry1Ab/c gene;
(2) utilize Primer Premier 5.0 software design PCR Auele Specific Primer and carry out blast checking;
(3) increase above-mentioned eight genes respectively;
(4) adopt fusion DNA vaccine method that eight gene splicings are become large fragment;
The first round reacts: with transgenic soybean lines GTS40-3-2, transgenic soybean lines A2704-12 with turn Bt cotton genomic dna for template, carries out pcr amplification with primer corresponding in claim 1; Reaction system is: cumulative volume is 50 μ l, wherein 10 times of Taq damping fluid 5 μ l, dNTPs (10mM) 4 μ l, each 4 μ l of upstream and downstream primer (10 μMs), and template (10ng/ μ l) 1 μ l, uses ddH
2o complements to 50 μ l.
Response procedures is: 95 DEG C of 5min; 30 circulations (94 DEG C of 30sec, 56.8-59.5 DEG C of 30sec, 72 DEG C of 30sec-60sec); 72 DEG C of 10min; 4 DEG C of preservations; Product Labeling is P
lec1, P
35S, P
nOS, P
pAT, P
35SG, P
cry, P
sad, P
ePS;
Second takes turns reaction, increases in two steps:
The first step: get each 70ng of first round reaction product, 10 times of Taq damping fluid 5 μ l, dNTPs (10mM) 4 μ l, uses ddH
2o complements to 50 μ l; Wherein P
lec1with P
35Sbe connected, P
nOSwith P
pATbe connected, P
35SG, P
cryand P
sadbe connected;
Response procedures: 15 circulations (94 DEG C of 20sec, 58.8-60 DEG C of 85sec), 4 DEG C of preservations;
Second step: add each 1 μ l of primer Lec-P1 and 35S-P2 each 1 μ l, primer Nos-P1 and Pat-P2 each 1 μ l, primer 35SG-P1 and Sad-P2 in the first step reaction solution respectively;
Response procedures: 94 DEG C of 3min, 28 circulations (94 DEG C of 15sec, 60 DEG C of 1min, 72 DEG C of 2min), 4 DEG C of preservations; P is labeled as by after fusion DNA vaccine product rubber tapping purifying
lec1-35S, P
nOS-PAT, P
35SG-Cry-Sad.
Third round is reacted: by product P
ePSand P
35SG-Cry-Sadcarry out fusion DNA vaccine reaction.
Get first round reaction product P
ePSthe product P of taking turns with second
35SG-Cry-Sadeach 2 μ l are as template, and primer is 35SG-P1 and Eps-P2 amplification; Reaction system is: cumulative volume 50 μ l, wherein 10 times of Taq damping fluid 5 μ l, dNTPs (10mM) 4 μ l, and each 1 μ l of upstream and downstream primer (10 μMs), uses ddH
2o complements to 50 μ l;
Response procedures: 94 DEG C of 3min, 28 circulations (94 DEG C of 15sec, 60 DEG C of 1min, 72 DEG C of 2min), 4 DEG C of preservations; By P
35SG-Cry-Sadwith P
ePSp is labeled as after connecting product rubber tapping purifying
35SG-Cry-Sad-EPS;
Fourth round is reacted, and increases in two steps:
The first step: get second take turns in reaction product P
lec1-35S, P
nOS-PATwith third round reaction product P
35SG-Cry-Sad-EPSeach 2 μ l work as template, and 10 times of Taq damping fluid 5 μ l, dNTPs (10mM) 4 μ l, uses ddH
2o complements to 50 μ l; 20 circulations (94 DEG C of 20sec, 72 DEG C of 30sec);
Second step: add Outside primer and Lec-P1 (10 μMs) and each 4 μ l of EPS-P2 (10 μMs) in the first step reaction solution, response procedures: 94 DEG C of 30sec, 28 circulations (94 DEG C of 30sec, 58.6 DEG C of 1min, 72 DEG C of 3min), 4 DEG C of preservations; Product Labeling is P
lE8.Reclaim purifying P
lE8, be connected to pMD
tM19-T Simple carrier, proceeds to intestinal bacteria TOP10 and can preserve use for a long time.
(5) large dna fragment cloning will be merged to pMD
tMon 19-T Simple carrier, screening obtains standard plasmid molecule pTLE8.
(6) the quantitative PCR detecting method checking of standard plasmid molecule pTLE8.
Described quantitative PCR detecting method checking, refer to the limit of detection of standard plasmid molecule pTLE8 when carrying out quantitative PCR analysis, quantitation limit and the characteristic such as repeatability and repeatability, to identify that this standard plasmid molecule replaces the ability of positive criteria material, and be applied to quantitative PCR detection transgenic soybean lines GTS-40-3-2 mensuration validity.
The beneficial outcomes that effective preparation genetically modified crops examination criteria molecule contrast Lectin1+35S+NOS+PAT+35SG+Sad+Cry1Ab/c+EPSPS method disclosed by the invention has is, utilizes fusion DNA vaccine to construct standard plasmid molecule pTLE8 containing eight genes first.This standard plasmid molecule can replace positive criteria material for the quantitative PCR detection of genetically engineered soybean, transgenic Bt cotton, solve the problem of the positives mark material want of testing process, without the need to providing multiple positive gene group DNA as positive control in testing process; This standard plasmid molecule is proceeded to intestinal bacteria, can steady in a long-termly preserve; New exogenous genetic fragment can be added, to meet the needs of increasing genetically modified crops material tests on the basis of original standard plasmid molecule.This standard plasmid molecule pTLE8 has that specificity is good, sensitivity advantages of higher in reality detects, and applies this standard plasmid molecule when carrying out actual sample quantitative analysis, in the allowed band that the deviation of sample detection result is extensively approved in the world.Therefore, the standard plasmid molecule pTLE8 built in the present invention is applicable to the quantitative analysis to the gm content in genetically engineered soybean, transgenic Bt cotton and converted products thereof completely.
Accompanying drawing explanation
The principle of Fig. 1 eight gene fusion PCR
A: first round PCR reacts: the single PCR of eight genes
B: the second takes turns PCR reaction: the mixing of contiguous gene with extend in advance
C: extend: the extension not adding primer
D:PCR reacts: the PCR reaction adding primer
Fig. 2 eight gene and each gel electrophoresis figure merging fragment PCR products
M1：DNA marker II；M2：DNA marker III；1：Lec1；2：35S；3：NOS；4：PAT；5：35SG；6：Cry；7：Sad1；8：EPSPS；9，14：LE8；10：Lec1+35S；11：NOS+PAT；12：35SG+Cry；13：35SG+Cry+Sad1+EPSPS
The sequencing result of Fig. 3 eight gene standard plasmid molecule pTLE8
Italic overstriking be fusion DNA vaccine primer position, be quantitative primer and probe position in square frame; Four restriction enzyme Not I, Sal I, EcoR I and Xba I are introduced between eight genes.
Embodiment
In order to explain enforcement of the present invention more fully, what provide genetically engineered soybean and cotton standard molecule contrast prepares embodiment.These embodiments are only explain instead of restriction point scope of invention.The experimental technique of actual conditions is not indicated, usually conveniently conditional operation in the following example.
Embodiment 1: the structure of standard plasmid molecule
One, experiment material
Transgenic soybean lines GTS40-3-2 and A2704-12; Non-transgenic soybean kind; Turn Bt cotton series.
Two, experiment reagent
PMD
tM19-T Simple Vector is purchased from Dalian Bao Bio-Engineering Company; DNTPs, Taq archaeal dna polymerase, DNA marker I, II and marker III are purchased from Beijing Quanshijin Biotechnology Co., Ltd; Restriction enzyme Not I, Sal I, EcoR I, Xba I are purchased from NEB Beijing company limited; The plasmid Mini Kit that plasmon DNA extraction and purifying adopt Beijing Tian Gen biochemical technology company limited to develop; Other biochemical reagents are import packing or domestic analytical pure.
Three, laboratory apparatus
TC-512 type PCR amplification instrument (TECHNE company of Britain); Geliance 200DNA running gel imager (PerkinElmer company of the U.S.); Nano-Drop ND-1000 nucleic acid-protein instrument (Nano Drop company of the U.S.); Whizzer; Thermostat water bath; Incubator; It equality.
Four, test method and process
1, plant genome DNA extracts-SDS method
A, take the soybean sample that 0.1g pulverizes through pulverizer, add the 2%SDS extract of 1ml 65 DEG C of preheatings, 10 μ l RNase-A mix, and in 65 DEG C of incubation 30min, are placed in the centrifugal 10min of 13000r after room temperature.
B, get the KAc resetting and adding 0.6 times of volume, after ice bath 20min, the centrifugal 10min of 13000r, gets and resets and add 2 times of volume precooling dehydrated alcohols, mixing.
C, in-20 DEG C of centrifugal 10min of standing 30min, 13000r, abandon supernatant, precipitation uses 70% washing with alcohol, abandons supernatant, repeat once after the short period of time is centrifugal.The molten precipitation of 200 μ l TE is added ,-20 DEG C of preservations after seasoning.
D, get 2 μ l extract DNA sample, with 1% agarose gel electrophoresis detection DNA quality.The DNA concentration and purity that utilize nucleic acid instrument to measure to extract.
2, design of primers
Soybean reference gene Lectin 1 is searched in GenBank, genetically modified crops screening sequence 35S and NOS, specificity PAT and EPSPS gene, the strain sequence of genetically engineered soybean GTS40-3-2, the foreign gene Cry1Ab/c of transgenic Bt cotton and reference gene Sad, software Primer 5.0 is utilized to design 5 pairs of qualitative PCR primers, be respectively used to amplification soybean reference gene specific sequence Lectin 1, screening sequence 35S and NOS, the exogenous insertion vector of specificity PAT and EPSPS gene and amplification genetically engineered soybean GTS40-3-2 and soybean gene group adjoining region specific sequence, cotton reference gene Sad and foreign gene Cry1Ab/c.Concrete primer sequence is in table 1.
Table 1 builds the PCR primer sequence of standard plasmid molecule
3, the independent pcr amplification of eight genes
According to the primer of table 1, with the genomic dna of transgenic soybean gene group DNA and transgene cotton Bt series for template, respectively pcr amplification is carried out to soybean reference gene Lectin 1,35S, NOS, PAT, EPSPS and GTS40-3-2 strain specificity sequence and cotton reference gene Sad, specific gene Cry.
Conveniently PCR method carries out the independent amplification of each object fragment.Amplification uses Taq archaeal dna polymerase, and reaction conditions and the amplified production length of each fragment amplification are as shown in table 2.The amplified production agarose gel electrophoresis of 1.5% detects, and determines object band, cuts glue Gel Extraction MiniKit and reclaim.Reduce the add-on of EB in gel preparation course, require EB concentration generally lower than 0.15 μ g/ml.
The reaction conditions of table 2 fragment amplification to be fused and amplified production length
4, fusion DNA vaccine reaction
Measure object fragment concentrations in each recovery product, equimolar fragment to be fused (amount of each gene to be fused is 70ng) is added in 50 μ l systems, do not add primer, the complementation using Taq archaeal dna polymerase to carry out each fragment extends, to form the fusion DNA vaccine product (intermediate product) of total length.Reaction conditions and intermediate product length as shown in table 3.
Table 3 fusion DNA vaccine reaction conditions and fusion product length
5, the total length pcr amplification of fragment is merged
Take intermediate product as template, in 50 μ l systems, add ddH successively
2o, 5 μ l 10 × PCR buffer, 4 μ l dNTPs 10mmol/L, 20 circulations (94 DEG C of 20sec, 72 DEG C of 30sec); Outside primer and Lec-P1 (10 μMs) and each 4 μ l of EPS-P2 (10 μMs) is added again, response procedures: 94 DEG C of 30sec, 28 circulations (94 DEG C of 30sec, 58.6 DEG C of 1min, 72 DEG C of 3min), 4 DEG C of preservations in reaction solution; The amplified production agarose gel electrophoresis of 1.0% detects, and determines object band, cuts glue Gel Extraction MiniKit and reclaim.
6, the clone of large fragment is merged
Utilize the method for molecular cloning that the fusion large fragment of eight genes is reclaimed product and be connected to pMD
tMon 19-T Simple carrier, linked system table 4.Transformation of E. coli TOP10 competent cell after 42 DEG C of heat shock 90s, blue hickie screens positive bacterium colony and bacterium colony PCR identifies, the positive colony order-checking that choosing qualification is correct.Extract its plasmid pTLE8 by after positive colony bacterium expanding propagation, and carry out double digestion (table 5) with Not I/Xba I, put 37 DEG C of water-bath 5-6 hour.
Table 4 merges the linked system of large fragment
Table 5 Not I/Xba I double digestion plasmid pMDLE8
7, pTLE8 standard plasmid molecule linearizing
The pTLE8 plasmid restriction endonuclease Sal I built or Eco R I carries out linearizing, as the standard molecule of genetically engineered soybean, cotton quantitative analysis.Gel reclaims linearizing plasmid DNA, with nucleic acid instrument measure linear plasmid DNA concentration.According to plasmid molecule size, the total mass number of plasmid can be converted into copy number.
Five, experimental result
1, the independent pcr amplification of fragment-eight gene to be fused in standard plasmid molecule
Conveniently PCR method carries out the independent amplification of each object fragment of Lec1,35S, NOS, PAT, EPSPS, 35SG, Cry and Sad, obtaining 530bp, 311bp, 253bp, 371bp, 369bp, 312bp, 335bp and 1199bp fragment respectively, is consistent (Fig. 2) with expection size.
2, fusion DNA vaccine reaction
Respectively Lec1 and 35S, NOS and PAT, G35, Cry and Sad are merged by bridging PCR, called after P
l35, P
nP, P
gS, size is respectively 841bp, 624bp and 1016bp, consistent with expection size.After will merging fragment purification, by P
gSwith P
ePScarry out the amplification of secondary fusion DNA vaccine, obtain the fusion fragment of 2215bp, called after P
gSE.By P
l35, P
nPand P
gSEcarry out third time fusion DNA vaccine amplification, the size obtained containing eight genes is the fusion large fragment (Fig. 1, Fig. 2) of 3680bp.
3, the clone, the enzyme that merge large fragment are cut and sequence verification
The fusion large fragment of above-mentioned eight genes is connected to plasmid vector pMD
tMon 19-T Simple Vector, select positive colony to check order, sequencing result is shown in accompanying drawing 3, containing Lec1,35S, NOS, PAT, G35, Cry, Sad and EPSPS eight genes, total length is 3680bp, and the homology of each gene order that eight genes log in GenBank is respectively 100%.This positive plasmid, after Not I/Xba I double digestion, has cut the object band that size is approximately 3680bp.Below all show that object fragment has successfully been cloned into pMD
tMon 19-T Simple Vector.
Embodiment 2: the application of standard plasmid molecule in reality detects of structure
One, enzyme and reagent
Real-time fluorescence quantitative PCR detection kit (Premix Ex Taq
tM(Perfect Real Time)) purchased from precious biotechnology (Dalian) company limited; Primer and TaqMan probe are synthesized by precious biotechnology (Dalian) company limited; Other biochemical reagents are import packing or domestic analytical pure.
Two, laboratory apparatus
Real-time fluorescence quantitative PCR instrument 7500 (ABI company).
Three, experimental technique and process
1, fluorescence quantification PCR primer and probe design synthesis
According to the sequence of Lectin in standard plasmid molecule 1 and EPSPS gene, software Primer Premier 5.0 is adopted to design quantification PCR primer and probe, in table 6.5 ' end of TaqMan probe marks reporter fluorescence dyestuff FAM and TET respectively, and 3 ' end marks cancellation non-fluorescence dyestuff BHQ and Eclipse respectively.Primer and probe synthesize by precious biotechnology (Dalian) company limited.Quantitative fluorescent PCR reaction system and reaction conditions are in table 7.
The quantitative primer of table 6 Lectin 1 and EPSPS gene and probe
Table 7 quantitative PCR reaction system and reaction conditions
2, the foundation of the typical curve of genetically engineered soybean GTS40-3-2 strain
Using linearization plasmid pTLE8 as standard DNA sample, be diluted to 3.66 × 10
5copy/μ l, 3.66 × 10
4copy/μ l, 3.66 × 10
3copy/μ l, 3.66 × 10
2copy/μ l, 3.66 × 10
1copy/μ l, production standard curve.
3, standard plasmid molecule pTLE8 is used for limit of detection (LOD) and the quantitation limit (LOQ) of quantitative PCR detection
Using the standard plasmid molecule DNA sample of different concns (as 40 copy/μ l, 20 copy/μ l, 10 copy/μ l, 6 copy/μ l) as LOD and LOQ of sample determination quantitative PCR detecting method.Each reaction in triplicate, according to the linear relationship between the typical curve of quantitative pcr amplification and amplification fluorescent signal, when determining to utilize the standard plasmid molecule pTLE8 replacement positive criteria material built, LOD and LOQ of genetically engineered soybean GTS40-3-2 structural specificity quantitative PCR detecting method.
4, the repeatability of standard plasmid molecule pTLE8 quantitative PCR detection system and repeatability
Optimize genetically engineered soybean GTS40-3-2 structural specificity quantitative PCR detection system and reaction conditions, respectively with the standard plasmid molecule genome DNA sample of different concns (as 3.66 × 10
5copy/μ l, 3.66 × 10
4copy/μ l, 3.66 × 10
3copy/μ l, 3.66 × 10
2copy/μ l, 3.66 × 10
1copy/μ l) carry out repeatability and circulation ratio test, each reaction is in triplicate.According to the typical curve of quantitative pcr amplification, determine repeatability and the repeatability of the genetically engineered soybean GTS40-3-2 structural specificity quantitative PCR reaction of setting up according to standard plasmid molecule pTLE8.
5, standard plasmid molecule pTLE8 quantitative PCR detection correction coefficient measures
When utilizing standard molecule to carry out quantitative analysis, must consider plasmid DNA and the efficiency variance of Soybean genomic DNA in PCR reaction, this difference can be passed through correction coefficient (Calibration Factors, Cf) and correct.The measuring method of Cf value sets up quantitative PCR detection system with standard plasmid molecule, carries out quantitative analysis to the genetically engineered soybean GTS40-3-2 standard positive material isozygotied, and calculated the correction coefficient Cf of standard plasmid molecule pTLE8 by the result comparing quantitative analysis.Cf value calculates by formula 1.After acquisition Cf value, the content obtaining transgene component in testing sample can be analyzed by formula 2.
The native gene copy number of the external source goal gene copy number that formula 1:Cf=isozygotys/isozygoty
Formula 2: percentage composition (%)=(sample external source goal gene copy number × 100)/(sample native gene copy number × Cf)
6, application standard plasmid molecule is to the quantitative analysis of genetically engineered soybean
According to correction coefficient and genetically engineered soybean Lec1, EPS gene quantification typical curve of the standard plasmid molecule pTLE8 measured, respectively 4 genetically engineered soybean reference material samples (gm content is respectively 2%, 1%, 0.5%, 0.1%) are analyzed, determine whether the standard plasmid molecule pTLE8 built can be effectively applied to the detection by quantitative of actual sample genetically engineered soybean.The accuracy of quantitative result is assessed by mean value and deviation, and accuracy is assessed by standard deviation and relative standard deviation.
Four, experimental result
1, the foundation of typical curve
Quantitative analysis method is set up as standard substance using linearization plasmid DNA, the amplification efficiency of the typical curve of Lec1 and EPS gene is respectively 99.416% and 100.164%, relation conefficient is respectively 0.996 and 0.999, explanation has good Ct value-starting point concentration dependency, can realize carrying out accurate quantitative analysis research to soybean sample.
2, the mensuration of detection by quantitative LOD, LOQ
Utilize the quantitative PCR reaction system optimized, respectively using the standard plasmid DNA sample of different concns (40 copy/μ l, 20 copy/μ l, 10 copy/μ l, 6 copy/μ l) as unknown sample, repeat experiment by 3 times, LOD and LOQ of quantitative PCR is respectively 9,15 copies.
3, quantitation curves repeatability is analyzed
Carry out reperformance test using the standard plasmid molecule DNA of different copy number as standard substance respectively, do three repetitions in the same way with condition, each three parallel.Repeatability result is as shown in table 8.The relative standard deviation of the Ct value obtained between parallel reactor and between different repetition is less than 3.85% (can accept within standard-required 35%), illustrate that the repeatability that quantitative PCR reacts is fine, there is very high stability, may be used for the further quantitative analysis of actual sample.
The repeatability analysis of table 8 quantitation curves
4, the mensuration of correction coefficient (Cf)
The pcr amplification efficiency caused due to the difference between plasmid DNA and plant genome DNA and the deviation of quantitative analysis results.When replacing genetically engineered soybean positive criteria material to be used for quantitative PCR detection with standard plasmid molecule DNA, correction coefficient (Cf) must be used to carry out correcting to eliminate deviation.Use the transgenic soybean DNA isozygotied to carry out calculation correction coefficient for template in experiment, the results are shown in Table 9, correction coefficient is 1.1.
The mensuration of table 9 standard plasmid molecule pMDLE8 quantitative PCR correction coefficient (Cf)
5, application standard plasmid molecule is to the quantitative analysis of genetically engineered soybean sample
According to correction coefficient and the quantitation curves of the standard plasmid molecule pTLE8 measured, respectively quantitative analysis is carried out to 4 transgenosis reference material samples.As can be seen from Table 10, the content of 4 reference material samples is respectively 2.01%, 1.18%, 0.66% and 0.35%, is respectively 1%, 14%, 16% and 10% with the deviation of actual value.Standard deviation is within the scope of 0.03-0.22, and relative standard deviation (can accept within 35%) within the scope of 3.09-18.53.As can be seen here, react the accurate quantification that can complete soybean sample with the quantitative PCR that plasmid DNA is set up, thus also illustrate that the plasmid pMDTE8 of this experimental construction can substitute the reference material of genome positive standard substance as soybean quantitative analysis completely.
The accuracy of the Quantitative PCR/Quantitative analysis of table 10 genetically engineered soybean and accuracy statistics
Claims (2)
1. a genetically engineered soybean, transgenic Bt cotton detection standard plasmid molecule, it is characterized in that, containing soybean native gene specific sequence Lectin 1, screening sequence 35S and NOS, the exogenous insertion vector of transgenic soybean lines A2704-12 transformation event pat gene specific sequence and transgenic soybean lines GTS40-3-2 transformation event EPSPS gene specific sequence and transgenic soybean lines GTS40-3-2 and the joining region sequence (35SG) of soybean gene group, cotton native gene Sad and transgene cotton exogenous fusion gene Cry1Ab/c, by fusion DNA vaccine, these 8 gene fusion are become a fragment, be inserted into carrier pMD
tMin 19-T Simple, obtain plasmid molecule pTLE8, merge fragment nucleotide sequence as SEQ ID No.17, its sequencing is Lectin1+35S+NOS+PAT+35SG+Sad+Cry1Ab/c+EPSPS, in standard molecule, the primer sequence of 8 genes and product size see the following form
。
2. genetically engineered soybean as claimed in claim 1, turn the construction process of Bt cotton detection standard plasmid molecule, it is characterized in that, comprise the following steps:
The first round reacts: with transgenic soybean lines GTS40-3-2, transgenic soybean lines A2704-12 with turn Bt cotton genomic dna for template, carries out pcr amplification with primer corresponding in claim 1; Reaction system is: cumulative volume is 50 μ l, wherein 10 times of Taq damping fluid 5 μ l, 10mM dNTPs4 μ l, and 10 μMs of upstream and downstream primer each 4 μ l, 10ng/ μ l template 1 μ l, use ddH
2o complements to 50 μ l;
Response procedures is: 95 DEG C of 5min; 30 circulations: 94 DEG C of 30sec, 56.8-59.5 DEG C of 30sec, 72 DEG C of 30sec-60sec; 72 DEG C of 10min; 4 DEG C of preservations; Product Labeling is P
lec1,p
35S, P
nOS, P
pAT, P
35SG, P
cry, P
sad, P
ePS;
Second takes turns reaction, increases in two steps:
The first step: get each 70ng of first round reaction product, 10 times of Taq damping fluid 5 μ l, 10mM dNTPs 4 μ l, uses ddH
2o complements to 50 μ l; Wherein P
lec1with P
35Sbe connected, P
nOSwith P
pATbe connected, P
35SG, P
cryand P
sadbe connected;
Response procedures: 10-15 circulation: 94 DEG C of 20sec, 58.8-60 DEG C of 85sec, 4 DEG C of preservations;
Second step: add each 1 μ l of primer Lec-P1 and 35S-P2 each 1 μ l, primer Nos-P1 and Pat-P2 each 1 μ l, primer 35SG-P1 and Sad-P2 in the first step reaction solution respectively;
Response procedures: 94 DEG C of 3min, 28 circulations: 94 DEG C of 15sec, 60 DEG C of 1min, 72 DEG C of 2min, 4 DEG C of preservations; P is labeled as by after fusion DNA vaccine product rubber tapping purifying
lec1-35S, P
nOS-PAT, P
35SG-Cry-Sad;
Third round is reacted: by product P
ePSand P
35SG-Cry-Sadcarry out fusion DNA vaccine reaction;
Get first round reaction product P
ePSproduct P in taking turns with second
35SG-Cry-Sadeach 2 μ l are as template, and primer is 35SG-P1 and Eps-P2 amplification; Reaction system is: cumulative volume 50 μ l, wherein 10 times of Taq damping fluid 5 μ l, 10mM dNTPs 4 μ l, and 10 μMs of each 1 μ l of upstream and downstream primer, use ddH
2o complements to 50 μ l;
Response procedures: 94 DEG C of 3min, 28 circulations: 94 DEG C of 15sec, 60 DEG C of 1min, 72 DEG C of 2min, 4 DEG C of preservations; By P
35SG-Cry-Sadwith P
ePSp is labeled as after connecting product rubber tapping purifying
35SG-Cry-Sad-EPS;
Fourth round is reacted, and increases in two steps:
The first step: get second and take turns middle the first step reaction product P
lec1-35S, P
nOS-PATwith third round reaction product P
35SG-Cry-Sad-EPSeach 2 μ l work as template, and 10 times of Taq damping fluid 5 μ l, 10mM dNTPs 4 μ l, uses ddH
2o complements to 50 μ l; 20 circulations: 94 DEG C of 20sec, 72 DEG C of 30sec;
Second step: add Outside primer i.e. 10 μMs of each 4 μ l of Lec-P1 and 10 μM EPS-P2 in the first step reaction solution, response procedures: 94 DEG C of 30sec, 28 circulations: 94 DEG C of 30sec, 58.6 DEG C of 1min, 72 DEG C of 3min, 4 DEG C of preservations; Product Labeling is P
lE8; Reclaim purifying P
lE8, be connected to pMD
tM19-T Simple carrier, proceeds to intestinal bacteria TOP10 and can preserve use for a long time.
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| CN104762310A (en) * | 2015-04-02 | 2015-07-08 | 贵州省烟草科学研究院 | High-coverage standard reference plasmid for detecting transgenic tobacco and application of high-coverage standard reference plasmid for detecting transgenic tobacco |
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| CN101063171A (en) * | 2007-05-24 | 2007-10-31 | 上海交通大学 | Standard plasmid molecule for detection of genetic improved soybean strain GTS40-3-2 and constructing method thereof |
| CN101724702A (en) * | 2009-12-23 | 2010-06-09 | 天津市农业科学院中心实验室 | Standard molecule contrast for genetically modified maize detection and prepration method thereof |
| CN101775440A (en) * | 2010-02-05 | 2010-07-14 | 吉林省农业科学院 | Plasmid control molecule for detection of transgenic soybean and building method thereof |
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| CN101063171A (en) * | 2007-05-24 | 2007-10-31 | 上海交通大学 | Standard plasmid molecule for detection of genetic improved soybean strain GTS40-3-2 and constructing method thereof |
| CN101724702A (en) * | 2009-12-23 | 2010-06-09 | 天津市农业科学院中心实验室 | Standard molecule contrast for genetically modified maize detection and prepration method thereof |
| CN101775440A (en) * | 2010-02-05 | 2010-07-14 | 吉林省农业科学院 | Plasmid control molecule for detection of transgenic soybean and building method thereof |
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| Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean;HIDEO KURIBAR;《Journal of AOAC International》;20021231;第85卷(第5期);第1077-1089页 * |
| 建立转基因作物及产品外源抗性基因检测技术体系的研究;曹际娟;《中国优秀硕博士学位论文全文数据库(博士)》;20041215(第4期);第41页第3.2.4节，第57-60页，尤其是第5.1节，第76-77页，第3.2.2节 * |
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