CN102409082B - Five-gene standard plasmid molecule for transgenic soybean detection and construction thereof - Google Patents
Five-gene standard plasmid molecule for transgenic soybean detection and construction thereof Download PDFInfo
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Abstract
The invention discloses a standard plasmid molecule necessary for transgenic crop detection and a construction method thereof, and particularly discloses a standard plasmid molecule for transgenic soybean detection and a construction method thereof. The standard plasmid molecule comprises a soybean endogenous gene Lectin-specific fragment, a 35S promoter and NOS terminator-specific fragment, a transgenic soybean line A2704-12 foreign gene PAT-specific fragment, and a transgenic soybean line GTS40-3-2 foreign gene EPSPS-specific fragment. By analyzing the specific sequences of the above five genes, specific PCR (polymerase chain reaction) primers are designed, and five target gene fragments are obtained by amplification. By a fusion PCR method, the five genes are fused to form a long fragment, and the long fragment is cloned to plasmid vector pMDTM19-T Simple to obtain a recombinant plasmid molecule pTLE5. The standard plasmid molecule constructed in the invention can completely substitute for a positive standard product, and is completely suitable for screening as well as structural-gene-specific qualitative and quantitative PCR analysis and detection of transgenic soybean lines GTS40-3-2 and A2704-12. If a new transgenic material emerges, a new foreign gene fragment can be added to an original standard molecule, thus the detection requirements of increasing transgenic crop materials are met.
Description
Technical field
What the present invention relates to is a kind of plasmid molecule of technical field of molecular biology, specifically, relates to a kind of genetically engineered soybean detection standard plasmid molecule and construction process thereof.
Background technology
Since nineteen ninety-six, whole world genetically modified crops cultivated area increases with the speed of average annual more than 10%, the genetically modified crops of whole world approval plantation in 2009 reach 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.Along with the extensive plantation of genetically modified crops, its safety issue causes the most attention of the global public.European Union signed first bill in the world in 1998, required to carry out label explanation to transgenic product; Within 1999, require that the non-transgenic product exporting to European Union must not pollute containing the transgenic product of 1%; 2002, the minimum limitation of mark was reduced to 0.9% by European Union.Thereupon, different regulations has been done to the minimum content of transgene component by the country such as Japan, Australia, Korea S, and threshold value is not from 1-5% etc.China has issued " agriculture GMO bio-safety management rules " May 23 calendar year 2001, on January 5th, 2002 discloses agriculture GMO bio-safety evaluation, mark and import security and manages three supporting management ways, determine the agriculture genetically modified organism catalogue that first implements identity management, and formal enforcement in 20 days March in 2002.
The enforcement of mark system depends on GMO detection technology.Mainly contain two class technology at present: a class is based on nuclei aoid methods, as PCR, gene chip etc., another kind of is based on method of protein, and as ELISA, test strip etc., wherein PCR method is most widely used.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.
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.In recent years, lot of domestic and foreign scholar reports some standard plasmid molecules for GMO detection.The capacity of standard plasmid molecule also has a large lifting, and the plasmid molecule from the plasmid molecule of initial simple target gene to multiple goal gene, these plasmid molecules can relate to multiple genes of the genetically modified crops of different plant species simultaneously.At present, 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 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.Utilize the PCR of the principle design of fusion DNA vaccine technology soybean Lectin gene, 35S promoter, NOS terminator, pat gene, EPSPS gene to merge primer, obtain the fusion large fragment of five 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 pTLE5.The standard plasmid molecule pTLE5 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.
Standard plasmid molecule of the present invention contains one section of sequence of soybean reference gene Lectin, 35S promoter, NOS terminator, pat gene, EPSPS gene, transgenic soybean lines GTS40-3-2 can be substituted and turn pat gene soybean, detecting for quantitative and qualitative analysis PCR.
A kind of genetically engineered soybean detection standard plasmid molecule of the present invention and construction process thereof, comprise the following steps:
(1) utilize GenBank database lookup and obtain soybean reference gene Lectin, 35S promoter, NOS terminator, EPSPS gene, pat gene;
(2) utilize Primer Premier 5.0 software design PCR Auele Specific Primer and carry out blast checking;
(3) increase above-mentioned five genes respectively;
(4) adopt fusion DNA vaccine method that five gene splicings are become large fragment;
The first round reacts: with transgenic soybean lines GTS40-3-2, transgenic soybean lines A2704-12 genomic dna for template, carry out pcr amplification with primer corresponding in claim 3; 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
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;
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 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.
Third round is reacted: by product P
lec1-35S, P
nOS-PATand P
ePScarry out fusion DNA vaccine reaction.
Get first round reaction product P
ePSproduct P in taking turns with second
lec1-35S, P
nOS-PATeach 2 μ l are as template, and primer is Lec-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 30sec, 28 circulations (94 DEG C of 15sec, 60 DEG C of 1min, 72 DEG C of 2min), 4 DEG C of preservations; By P
lec1-35S, P
nOS-PATand P
ePSconnection Product Labeling is P
lE5.
(5) purifying P is reclaimed
lE5, be connected to pMD
tM19-T Simple carrier, proceeds to intestinal bacteria TOP10 screening and obtains standard plasmid molecule pTLE5.
(6) the qualitative PCR detection method checking of standard plasmid molecule pTLE5.
(7) the quantitative PCR detecting method checking of standard plasmid molecule pTLE5.
Described qualitative PCR checking, refer in the standard plasmid molecule pTLE5 built with qualitative PCR methods analyst and can amplify the soybean native gene Lectin 1 identical with transgenic soybean lines GTS-40-3-2 with A2704-12 and the specific sequence of 35S, NOS, EPSPS and pat gene, and the requirement of qualitative PCR detection can be met to the detection sensitivity of specific gene in pTLE5.
Described quantitative PCR checking, refer to the limit of detection of soybean native gene Lectin 1 and transgenic soybean lines GTS-40-3-2 foreign gene EPSPS specific sequence in the standard plasmid molecule pTLE5 built with quantifying PCR method 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 Lectin 1+35S+NOS+PAT+EPSPS method disclosed by the invention has is, utilizes fusion DNA vaccine to construct standard plasmid molecule pTLE5 containing five genes first.This standard plasmid molecule can replace positive criteria material for the quantitative PCR detection of genetically engineered soybean, thus solves 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; In addition, also new exogenous genetic fragment can be added on the basis of original standard plasmid molecule, to meet the needs of increasing genetically modified crops material tests.This standard plasmid molecule pTLE5 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 pTLE5 built in the present invention is applicable to the quantitative analysis to the gm content in genetically engineered soybean and converted products thereof completely.
Accompanying drawing explanation
Fig. 1 five 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：EPSPS；6：Lec1+35S；7：NOS+PAT；8：LE5
The structure schematic diagram of Fig. 2 five gene standard plasmid molecule pTLE5 and sequencing result
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 standard molecule contrast below 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; Transgenic cotton flower variety.
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 200 DNA 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 native gene Lectin 1, genetically engineered soybean screening sequence 35S and NOS, genetically engineered soybean foreign gene PAT and EPSPS gene order is searched in GenBank, utilize software Primer 5.0 to design 5 pairs of qualitative PCR primers, be respectively used to amplification soybean reference gene specific sequence Lectin 1, genetically engineered soybean screening sequence 35S and NOS, genetically engineered soybean foreign gene PAT and EPSPS gene specific sequence.Concrete primer sequence is in table 1.
Table 1 builds the PCR primer sequence of standard plasmid molecule
3, the independent pcr amplification of five genes
According to the primer of table 1, with transgenic soybean gene group DNA for template, respectively pcr amplification is carried out to the 35S promoter of soybean native gene Lectin 1, genetically engineered soybean and NOS terminator, genetically engineered soybean foreign gene PAT and EPSPS gene.
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 ddH2O successively, 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 in 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; 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
Utilizing the method for molecular cloning that the fusion large fragment of five genes is reclaimed product is connected on pMDTM19-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 carries out bacterium colony PCR qualification, the positive colony order-checking that choosing qualification is correct.Extract its plasmid pTLE5 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 3-4 hour.
Table 4 merges the linked system of fragment
Reaction reagent volume (μ l)
Solution Ⅰ 5.0
Pcr amplification product 4.2
PMD 19-T Simple carrier 0.8
Table 5Not I/Xba I double digestion plasmid pMDLE8
Reaction reagent volume (μ l)
buffer 2
pTLE5 5
Not I/Xba I each 1
100％BSA 0.2
H
2O 4.8
7, pTLE5 standard plasmid molecule linearizing
The pTLE5 plasmid restriction endonuclease Sal I built or EcoR I carries out linearizing, as the standard molecule of genetically engineered soybean 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-five 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 and EPSPS, obtains 530bp, 311bp, 253bp, 383bp and 1199bp fragment respectively, is consistent (Fig. 2) with expection size.
2, fusion DNA vaccine reaction
Lec1 and 35S, NOS and PAT are merged respectively by bridging PCR, called after L35, NP, size is respectively 841bp and 636bp, consistent with expection size.After will merging fragment purification, L35, NP and EPSPS are carried out the amplification of second time fusion DNA vaccine, the size obtained containing five genes is the fusion large fragment (Fig. 2) of 2676bp.
3, the clone, the enzyme that merge large fragment are cut and sequence verification
The fusion large fragment of above-mentioned five 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 1, containing Lec1,35S, NOS, PAT and EPSPS five genes, total length is 2676bp, and the homology of each gene order that five 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 2663bp.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, the qualitative PCR of standard plasmid molecule pTLE5 detects
By consulting pertinent literature, the qualitative PCR detection method of acquisition soybean reference gene Lectin 1, genetically engineered soybean GTS40-3-2 and A2704-12 structure gene specificity, genetically engineered soybean screening sequence 35S promoter and NOS terminator.PCR reaction system is: cumulative volume is 50 μ l, wherein Taq damping fluid (10 ×) 5 μ l, and each 2 μ l of upstream and downstream primer (10 μMs), DNA profiling 1 μ l, complements to 50 μ l with ddH2O.
Response procedures is: 95 DEG C of 3min; 30cycles (95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min); 72 DEG C of 10min, 4 DEG C of preservations.
Be that template carries out qualitative PCR detection with the DNA of plasmid molecule pTLE5 (6830 copy/μ l), genetically engineered soybean GTS40-3-2 and A2704-12, Non-transgenic soybean and other transgenic lines, judge the specificity that plasmid molecule is used for qualitative PCR and detects.
Carry out qualitative PCR amplification with the DNA sample of the plasmid molecule pTLE5 of different concns (68300,6830,683,68.3,6.83 copy/μ l) for template, judge that plasmid molecule is used for the sensitivity of qualitative PCR detection.
2, 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
3, the foundation of the typical curve of genetically engineered soybean GTS40-3-2 strain
Using linearization plasmid pTLE5 as standard DNA sample, be diluted to 5.4 × 10
5copy/μ l, 5.4 × 10
4copy/μ l, 5.4 × 10
3copy/μ l, 5.4 × 10
2copy/μ l, 5.4 × 10
1copy/μ l, production standard curve.
4, standard plasmid molecule pTLE5 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 pTLE5 replacement positive criteria material built, LOD and LOQ of genetically engineered soybean GTS40-3-2 structural specificity quantitative PCR detecting method.
5, standard plasmid molecule pTLE5 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 pTLE5 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 pTLE5 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 pTLE5 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 qualitative PCR of standard plasmid molecule pTLE5 detects
Plasmid molecule pTLE5 (6830 copy/μ l), genetically engineered soybean GTS40-3-2 and A2704-12, Non-transgenic soybean and transgenic cotton floral material are carried out to the result display of qualitative PCR detection, in pTLE5 and GTS40-3-2, all can amplify Lectin 1,35S, NOS and EPSPS gene.Lectin 1,35S, NOS and pat gene is amplified in pTLE5 and A2704-12.Lectin 1 gene can only be amplified in Non-transgenic soybean, and do not amplify spawn in transgene cotton.Above result shows that plasmid molecule pTLE5 and genetically engineered soybean GTS40-3-2, A2704-12 positive criteria material have the specificity of equivalence.
With the DNA sample of the plasmid molecule pTLE5 of different concns (68300,6830,683,68.3,6.83 copy/μ l) for template carries out qualitative PCR amplification, result shows, plasmid molecule pTLE5 concentration 68.3 copy/μ l and above time sample all can be stable amplify five genes (Lectin 1,35S, NOS, PAT and EPSPS), show that the qualitative PCR detection sensitivity of plasmid molecule pTLE5 can reach 68.3 copies.
These results suggest that plasmid molecule pTLE5 of the present invention can substitute genetically engineered soybean GTS40-3-2 and A2704-12 positive criteria material, the qualitative PCR for genetically engineered soybean GTS40-3-2 and A2704-12 detects.
2, 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 all greater than 0.99, illustrates to have good linear relationship, can realize carrying out accurate quantitative analysis research to soybean sample.
3, 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.
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 8 standard plasmid molecule pTLE5 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 pTLE5 measured, respectively quantitative analysis is carried out to 4 transgenosis reference material samples.As can be seen from Table 9, 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 pTLE5 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 9 genetically engineered soybean and accuracy statistics
Claims (3)
1. a genetically engineered soybean detection standard plasmid molecule, it is characterized in that, from transgenic soybean gene group DNA, soybean native gene Lectin 1, screening sequence 35S promoter and NOS terminator, foreign gene PAT and EPSPS gene is amplified by Specific PCR primers, utilize fusion DNA vaccine that these five gene fusion are become a fragment, and be once building up to plasmid vector pMD
tMin 19-T Simple, obtain standard plasmid molecule pTLE5, its molecular size is 5368bp; In standard molecule the primer sequence of 5 genes and product size as follows:
。
2. genetically engineered soybean detection standard plasmid molecule according to claim 1, it is characterized in that described fusion fragment nucleotide sequence is as SEQ ID No.11, its sequencing is Lectin 1+35S+NOS+PAT+EPSPS, and size is 2676bp.
3. a construction process for genetically engineered soybean detection standard plasmid molecule as claimed in claim 1, is characterized in that, comprise the following steps:
(1) utilize biological data storehouse to carry out bioinformatic analysis, obtain the specific sequence of soybean native gene Lectin 1, genetically engineered soybean screening sequence 35S and NOS, transgenic soybean lines A2704-12 foreign gene PAT and transgenic soybean lines GTS40-3-2 foreign gene EPSPS;
(2) PCR Auele Specific Primer is designed;
(3) fusion DNA vaccine of Lectin 1+35S+NOS+PAT+EPSPS gene:
The first round reacts: with transgenic soybean lines GTS40-3-2, transgenic soybean lines A2704-12 genomic dna for template, carry 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 30 ~ 60sec; 72 DEG C of 10min; 4 DEG C of preservations; Product Labeling is P
lecl, P
35S, P
nOS, P
pAT, 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
leclwith P
35Sbe connected, P
nOSwith P
pATbe 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 1 of primer Lec-P1 and 35S-P2 each 1 μ l, primer Nos-P1 and Pat-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
lecl-35S, P
nOS-PAT;
Third round is reacted: by product P
lecl-35S, P
nOS-PATand P
ePScarry out fusion DNA vaccine reaction;
Get first round reaction product P
ePSproduct P in taking turns with second
lecl-35S, P
nOS-PATeach 2 μ l are as template, and primer is Lec-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 30sec, 28 circulations: 94 DEG C of 15sec, 60 DEG C of 1min, 72 DEG C of 2min, 4 DEG C of preservations; By P
lecl-35S, P
nOS-PATand P
ePSconnection Product Labeling is P
lE5;
(4) purifying P is reclaimed
lE5, be connected to pMD
tM19-T Simple carrier, obtains standard plasmid molecule pTLE5.
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| 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 |
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| 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 |
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