CN102154454B - Method for detecting transgenosis constituents in plant by GeXP multi-PCR (polymerase chain reaction) technology and application thereof - Google Patents
Method for detecting transgenosis constituents in plant by GeXP multi-PCR (polymerase chain reaction) technology and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting transgenosis constituents in plant by a GeXP multi-PCR (polymerase chain reaction) technology and application thereof. The method comprises the following steps of: performing the PCR and the capillary electrophoresis by designing the primer of the common exogenous gene such as 5-shikimic acid-3-phosphoric acid synzyme gene, glufosinate acetyl transferase gene, glufosinate acetyl transferase gene, monomeric Mosaic virus 35S promoter, cauliflower mosaic virus 35S promoter and nopaline synzyme gene terminator, and analyzing according to the result of the capillary electrophoresis, wherein the PCR is used for triggering the target genes to be amplified by the common primers. The amplification preference during multi-PCR can be effectively solved, and the detection sensitivity can be improved due to the PCR; the resolution ratio can be improved due to the capillary electrophoresis; and the result can be judged according to the length of the amplified product segment, and the detection specificity can be preferably improved. The invention provides a method for detecting the transgenosis plant more reliably and conveniently, so that the built system is high in repeatability, and the multiple tests are good in stability.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of GeXP (GenomeLab eXpress Profiling) multiple PCR technique detects method and the application of transgene component in the plant.
Background technology
Along with the increasing and global cultivated area of genetically modified crops kind constantly enlarges, genetically modified food has become the important component part in global food consumption market.The security of genetically modified food is edible safety especially, also causes people's concern day by day.Along with the development of international trade and transgenic technology, European Union and the U.S., Japan and other countries have successively been put into effect corresponding law and management process, genetically modified food is carried out forced sign or voluntary sign.China promulgates " agriculture genetically modified organism security control regulations " May 23 calendar year 2001, " the agriculture genetically modified organism identity management way " that came into effect on March 20th, 2002 regulation, and country carries out inspection and quarantine and sign system to agriculture genetically modified organism.Can see, methods for detecting transgenic foods has been become inevitable world trend.Therefore we must strengthen the further investigation to the genetically modified food detection technique.The screening of some foreign gene Chang Zuowei genetically modified crops detects gene, as 5-shikimic acid-3-phosphate synthase gene (EPSPS), derive from bacillus grass fourth phosphinothricin acetyl transferase gene (BAR), grass fourth phosphinothricin acetyl transferase gene (PAT), radix scrophulariae mosaic virus 35 S promoter (FMV35S), cauliflower mosaic virus 35S promoter (CaMV35S), rouge alkali synthetase gene terminator (NOS).
At present, more to transgenic crop detection technique kind both at home and abroad, be divided into two kinds substantially: the one, detect the protein that whether contains external source, namely the expression of exogenous gene product mainly adopts the ELISA method; The 2nd, detect and whether contain foreign gene (DNA), mainly contain gene chips and PCR detection method, wherein round pcr is comparatively ripe, has characteristics such as quick, easy, sensitivity.At present, domestic and international many R﹠D institutions and associated mechanisms are applied to the genetically modified crops detection with the substance round pcr.The main limitation of substance round pcr is that flux is not high, this is because single-gene PCR can only detect a gene or transgenosis element at every turn, if detect foreign gene, transgenosis element or the evaluation foreign gene in the transgenic plant, the kind of transgenosis element etc. simultaneously, need be a plurality of single PCR, and need know fragments sequence information to be checked, complex operation not only, time-consuming and cost is higher, and the transgenosis component kind is many in actual applications, sequence is uncertain, is difficult to detect one by one and identify.Multiplex PCR has solved the problems referred to above to a certain extent, can one-time detection and identify a plurality of foreign genes or transgenosis element, improved detection efficiency.But also there are some general problems in multiple PCR method itself, and is not high as specificity and sensitivity; Several to primer owing in the multi-PRC reaction system, existing simultaneously, make the probability that forms complicated primer dimer increase greatly, reaction power is also extremely complicated; Certain several particular patch section preferentially increases in the amplification, and other fragments can not amplify; The goal gene quantity of Jian Ceing is generally less than 10 grades (how at 3-5 gene) simultaneously, makes multiplex PCR also not reach the target of high-throughput rapid detection and analysis.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of GeXP multiple PCR technique to detect the method for transgene component in the plant with not enough.This method can detect the multiple transgene component that changes in the plant simultaneously.
Another object of the present invention is to provide described GeXP multiple PCR technique to detect the application of the method for transgene component in the plant.
Purpose of the present invention is achieved through the following technical solutions: a kind of GeXP multiple PCR technique detects the method for transgene component in the plant, may further comprise the steps:
(1) primer of design foreign gene and native gene, following primer is 5 '-3 ';
The upstream and downstream primer of foreign gene 5-shikimic acid-3-phosphate synthase gene (EPSPS) is respectively:
EPSPS-F：AGGTGACACTATAGAATAGTTGCGGCCCTGCTTGTT
EPSPS-R：GTACGACTCACTATAGGGAGGCGGTCGCTTTCCTTGA；
The upstream and downstream primer of foreign gene cauliflower mosaic virus 35S promoter (CaMV35S) is respectively:
CaMV35s-F：AGGTGACACTATAGAATAGCTCCTACAAATGCCATCA
CaMV35s-R：GTACGACTCACTATAGGGAGATAGTGGGATTGTGCGTCA；
The upstream and downstream primer of foreign gene rouge alkali synthetase gene terminator (NOS) is respectively:
NOS-F：AGGTGACACTATAGAATAATCGTTCAAACATTTGGCA
NOS-R：GTACGACTCACTATAGGGAATTGCGGGACTCTAATCATA；
The upstream and downstream primer of foreign gene grass fourth phosphinothricin acetyl transferase gene (BAR) (deriving from bacillus Bacillus amyloliquefaciens) is respectively:
Bar-F：AGGTGACACTATAGAATATCTGCACCATCGTCAACCACT
Bar-R：GTACGACTCACTATAGGGACTCCAGGGACTTCAGCAGGTG；
The upstream and downstream primer of foreign gene grass fourth phosphinothricin acetyl transferase gene (PAT) is respectively:
PAT-F：AGGTGACACTATAGAATACGGAGAGGAGACCAGTTGAG
PAT-R：GTACGACTCACTATAGGGATGGGTGTTTGTGGCTCTGT；
The upstream and downstream primer of foreign gene radix scrophulariae mosaic virus 35 S promoter (FMV35S) is respectively:
FMV35S-F：AGGTGACACTATAGAATAAAGACATCCACCGAAGACTTA
FMV35S-R：GTACGACTCACTATAGGGAAGGACAGCTCTTTTCCACGTT；
The upstream and downstream primer of native gene phosphoric acid enol pyruvic acid carboxylase gene (PEP) is respectively:
PEP-F：AGGTGACACTATAGAATAGCTAGTGTAGACCAGTTCTTG
PEP-R：GTACGACTCACTATAGGGACACTCTTGTCTCTTGTCCTC；
The upstream and downstream primer of native gene plant soybean lectin plain gene (Lectin) is respectively:
Lectin-F：AGGTGACACTATAGAATAGCCCTCTACTCCACCCCCATCC
Lectin-R：GTACGACTCACTATAGGGAGCCCATCTGCAAGCCTTTTTGTG；
The upstream universal primer is: GF:AGGTGACACTATAGAATA; Wherein this primer carries out fluorescent mark;
The downstream universal primer is: GR:GTACGACTCACTATAGGGA; Wherein this primer carries out fluorescent mark;
The preferred fluorescein Cy5 of described fluorescent mark;
(2) genome of extraction sample;
(3) carry out multiplex PCR: the genome that obtains with step (2) is template, the primer of foreign gene and native gene described in the step (1) can independent assortment, described upstream universal primer and downstream universal primer are essential primer, carry out PCR, obtain the PCR product;
(4) capillary electrophoresis detects the PCR product;
(5) result is analyzed.
PCR described in the step (3) more preferably according to the kind difference of sample, selects primer:
1. when sample is soybean, primer be EPSPS-F, EPSPS-R, CaMV35s-F, CaMV35s-R, NOS-F, NOS-R, Lectin-F and Lectin-R by etc. the primer A that obtains of mixed in molar ratio, and upstream universal primer and downstream universal primer by etc. the primer D that obtains of mixed in molar ratio;
2. when sample is vegetable seed, primer be Bar-F, Bar-R, PAT-F, PAT-R, FMV35S-F, FMV35S-R, NOS-F, NOS-R, PEP-F and PEP-R by etc. the primer B that obtains of mixed in molar ratio, and upstream universal primer and downstream universal primer by etc. the primer D that obtains of mixed in molar ratio;
3. when sample is soybean and vegetable seed mixture, primer be EPSPS-F, EPSPS-R, CaMV35s-F, CaMV35s-R, Bar-F, Bar-R, PAT-F, PAT-R, FMV35S-F, FMV35S-R, NOS-F and NOS-R by etc. the primer C that obtains of mixed in molar ratio, and upstream universal primer and downstream universal primer by etc. the primer D that obtains of mixed in molar ratio;
The reaction system of described PC R is preferably: per 25 μ l PCR reaction systems contain: primer A, primer B or primer C 1.25pmol, primer D 12.5pmol, genome 20～500ng;
The reaction conditions of described PC R is preferably 95 ℃ of 10min; 95 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 30s, 35 circulations; 72 ℃ of 10min;
The operation steps of capillary electrophoresis is preferably described in the step (4): use sample solution (SLS on the sample, Sample Loading Solution) and DSS (DNA Size Standard)-400 155: 1 by volume thorough mixings, in each hole of last model, add the aforementioned mixed solution of 39 μ l then, getting the PCR product that 1 μ l step (3) obtains again adds wherein, blow and beat 3～4 times, cover a dropstone wax oil at last in every hole; Every hole adds dissociating buffer, carries out capillary electrophoresis;
The add-on of described dissociating buffer is preferably 250 μ l;
The condition of capillary electrophoresis is described in the step (4):
Kapillary: 50 ℃ of temperature;
Sex change: 90 ℃, 120sec;
Inject sample: 2.0Kv, 30sec;
Separate: 6.0Kv, 35min;
Interpretation of result described in the step (5) is to judge gene according to X-coordinate size bp numerical values recited, wherein: Lectin 155bp, NOS 202bp, CaMV35s 232bp, EPSPS 353bp, Bar 321bp, PAT 161bp, FMV35S 247bp, PEP 285bp.
The method of transgene component is applied to the detection of transgene component in the plant in the described GeXP multiple PCR technique detection plant, particularly is applied to the detection of transgene component in soybean and the vegetable seed.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention causes the target gene amplification by adopting universal primer, can effectively solve the amplification preferences in the multiplex PCR process, and then adopts capillary electrophoresis and fluorescent mark amplified production to improve detection sensitivity and resolving power; According to amplified production fragment length judged result, help to improve detection specificity.
(2) the present invention provides comparatively reliable and easy detection method for the detection of transgenic plant, and the system of setting up is repeatable high, through the test of many times good stability.Its detected result is compared with other detection techniques, has bigger reliability and adaptability, has simplified the detection step, has improved detection efficiency, thereby the detection means of more promising effect is provided for the detection of transgenic plant.
Description of drawings
Fig. 1 utilizes the method for the invention to detect the figure as a result that genetically engineered soybean obtains.
Fig. 2 utilizes the method for the invention to detect the figure as a result that the transgenosis vegetable seed obtains.
Fig. 3 utilizes the method for the invention to detect the figure as a result of six foreign genes of genetically engineered soybean and transgenosis vegetable seed mixture gained.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Embodiment 1
(1) genomic dna of extracting sample
Get genetically engineered soybean (RRS strain national standard sample XLSY-ZJY-007, Beijing Xing Linsheng industry development in science and technology center) after crushed, specification sheets according to the Wizard Genomic DNA Purification Kit of Promega company extracts genomic dna, measure the ultraviolet light absorption value of its 260nm and 280nm, calculate concentration and the purity of the DNA that extracts.According to the concentration of surveying dna solution is diluted to 100ng/ μ l and place-20 ℃ standby.
(2) integrity of detection genomic dna
Endogenous reference gene plant soybean lectin plain gene (Lectin) by the amplification soybean carries out the PCR reaction, the reaction system of PCR is 25 μ l, wherein contain: Ex Taq12.5 μ l, each (1 μ mol/L) 2.5 μ l of endogenous reference gene upstream and downstream primer (being Lectin-F and Lectin-R), dna profiling 1 μ l, the sterilization distilled water complements to 25 μ l.The reaction conditions of PCR is: 95 ℃, and 10min; (95 ℃ of 30s; 58 ℃ of 1min; 72 ℃ of 30s) * 35 circulation; 72 ℃ of 10min.
PCR result confirms that the genomic dna that extracts can be used as the template of GeXP multiple PCR technique.
(3) GeXP multiple PCR technique
Following primer (being 5 '-3 ') by waiting mole number to mix, is obtained primer A:
The upstream and downstream primer of foreign gene 5-shikimic acid-3-phosphate synthase gene (EPSPS) is respectively:
EPSPS-F：AGGTGACACTATAGAATAGTTGCGGCCCTGCTTGTT
EPSPS-R：GTACGACTCACTATAGGGAGGCGGTCGCTTTCCTTGA；
The upstream and downstream primer of foreign gene cauliflower mosaic virus 35S promoter (CaMV35S) is respectively:
CaMV35s-F?AGGTGACACTATAGAATAGCTCCTACAAATGCCATCA
CaMV35s-R：GTACGACTCACTATAGGGAGATAGTGGGATTGTGCGTCA；
The upstream and downstream primer of foreign gene rouge alkali synthetase gene terminator (NOS) is respectively:
NOS-F：AGGTGACACTATAGAATAATCGTTCAAACATTTGGCA
NOS-R：GTACGACTCACTATAGGGAATTGCGGGACTCTAATCATA；
The upstream and downstream primer of native gene plant soybean lectin plain gene (Lectin) is respectively:
Lectin-F：AGGTGACACTATAGAATAGCCCTCTACTCCACCCCCATCC
Lectin-R：GTACGACTCACTATAGGGAGCCCATCTGCAAGCCTTTTTGTG；
Following primer by waiting mole number to mix, is obtained primer D:
Upstream universal primer sequence: GF:Cy5-AGGTGACACTATAGAATA
Downstream universal primer sequence: GR:Cy5-GTACGACTCACTATAGGGA.
Get 200 μ L PCR pipe, add Ex Taq (Takara) 12.5 μ l, last, primer A (1 μ mol/L) 1.25 μ l, primer D (10 μ mol/L) 1.25 μ l, dna profiling 1 μ l, the sterilization distilled water complements to 25 μ l.The PCR reaction tubes is put into the PCR instrument, and amplification condition is 95 ℃ of 10min; 95 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 30s, 35 circulations; 72 ℃, 10min.
Extract reaction solution 1 μ l and carry out capillary electrophoresis:
The system of capillary electrophoresis is 40 μ l:
SLS (Sample Loading Solution) and DSS (DNA Size Standard)-400 155: 1 by volume thorough mixings obtain mixed solution H; At each Kong Zhongjun packing 39 μ l mixed solution H of last model, get the PCR product that 1 μ l step (3) obtains and add wherein, blow and beat 3～4 times, cover a dropstone wax oil at last in every hole; Every hole adds dissociating buffer, carries out capillary electrophoresis.SLS, DSS-400 and dissociating buffer are all available from U.S. Beckman Coulter Inc..
The condition of capillary electrophoresis is:
Kapillary: 50 ℃ of temperature;
Sex change: 90 ℃, 120sec;
Inject sample: 2.0Kv, 30sec;
Separate: 6.0Kv, 35min;
This goes on foot used instrument: Beckman Genomelab Gexp-Genetic Analysis System.
As seen the result has 4 peaks as shown in Figure 1.Judge gene according to X-coordinate size bp numerical values recited, wherein: Lectin 155bp, NOS 202bp, CaMV35s 232bp, EPSPS 353bp.From left to right be respectively Lectin, NOS, CaMV 35S, EPSPS gene.As seen method provided by the present invention can detect transgene component exactly from soybean.
Embodiment 2
(1) genomic dna of extracting sample
(RT73 is national standard sample XLSY-ZJY-005 to get transgenic rapeseed, Beijing Xing Linsheng industry development in science and technology center) after crushed, Wizard Genomic DNA Purification Kit according to Promega company extracts DNA, measure the ultraviolet light absorption value of its 260nm and 280nm, calculate concentration and the purity of the DNA that extracts.According to the concentration of surveying dna solution is diluted to 100ng/ μ l and place-20 ℃ standby.
(2) integrity of detection genomic dna
The transgenosis vegetable seed of preparation confirms can be used as the pcr amplification template by the endogenous reference gene phosphoric acid enol pyruvic acid carboxylase gene (PEP) of amplification vegetable seed.The reaction system of PCR and reaction conditions are with embodiment 1 step (2), and difference only is to change primer into PEP-F and PEP-R.
(3) GeXP multiple PCR technique
Following primer by waiting mole number to mix, is obtained primer B:
The upstream and downstream primer of foreign gene rouge alkali synthetase gene terminator (NOS) is respectively:
NOS-F：AGGTGACACTATAGAATAATCGTTCAAACATTTGGCA
NOS-R：GTACGACTCACTATAGGGAATTGCGGGACTCTAATCATA；
The upstream and downstream primer of foreign gene bacillus grass fourth phosphinothricin acetyl transferase gene (BAR) is respectively:
Bar-F：AGGTGACACTATAGAATATCTGCACCATCGTCAACCACT
Bar-R：GTACGACTCACTATAGGGACTCCAGGGACTTCAGCAGGTG；
The upstream and downstream primer of foreign gene grass fourth phosphinothricin acetyl transferase gene (PAT) is respectively:
PAT-F：AGGTGACACTATAGAATACGGAGAGGAGACCAGTTGAG
PAT-R：GTACGACTCACTATAGGGATGGGTGTTTGTGGCTCTGT；
The upstream and downstream primer of foreign gene radix scrophulariae mosaic virus 35 S promoter (FMV35S) is respectively:
FMV35S-F：AGGTGACACTATAGAATAAAGACATCCACCGAAGACTTA
FMV35S-R：GTACGACTCACTATAGGGAAGGACAGCTCTTTTCCACGTT；
The upstream and downstream primer of native gene phosphoric acid enol pyruvic acid carboxylase gene (PEP) is respectively:
PEP-F：AGGTGACACTATAGAATAGCTAGTGTAGACCAGTTCTTG
PEP-R：GTACGACTCACTATAGGGACACTCTTGTCTCTTGTCCTC；
Following primer by waiting mole number to mix, is obtained primer D:
Upstream universal primer sequence: GF:Cy5-AGGTGACACTATAGAATA
Downstream universal primer sequence: GR:Cy5-GTACGACTCACTATAGGGA;
Get 200 μ L PCR pipe, add Ex Taq (Takara) 12.5 μ l, last, primer B (1 μ mol/L) 1.25 μ l, primer D (10 μ mol/L) 1.25 μ l, dna profiling 1 μ l, the sterilization distilled water complements to 25 μ l.The PCR reaction tubes is put into the PCR instrument, and amplification condition is 95 ℃ of 10min; 95 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 30s, 35 circulations; 72 ℃, 10min.
Extract reaction solution 1 μ l and carry out capillary electrophoresis:
The system of capillary electrophoresis is 40 μ l:
Behind SLS (Sample Loading Solution) and DSS (DNA Size Standard)-400 155: 1 by volume thorough mixings of ratio at this mixed solution of each Kong Zhongjun packing 39 μ l of last model, getting the PCR product that 1 μ l step (3) obtains adds wherein, blow and beat 3～4 times, cover a dropstone wax oil at last in every hole; Every hole adds dissociating buffer, carries out capillary electrophoresis.
The condition of capillary electrophoresis is:
Kapillary: 50 ℃ of temperature
Sex change: 90 ℃, 120sec
Inject sample: 2.0Kv, 30sec
Separate: 6.0Kv, 35min
This goes on foot used instrument: Beckman Genomelab Gexp-Genetic Analysis System.
The result can clearly observe 5 peaks as shown in Figure 2, judges gene according to X-coordinate size bp numerical values recited, wherein: NOS 202bp, Bar 321bp, PAT 161bp, FMV35S 247bp, PEP 285bp.From left to right be respectively PAT, NOS, FMV35S, PEP and Bar gene.As seen method provided by the present invention can detect transgene component exactly from vegetable seed.
Embodiment 3
(1) genomic dna of extracting sample
Get genetically engineered soybean and transgenic rapeseed after crushed, according to the WizardGenomic DNAPurification Kit extraction DNA of Promega company, measure the ultraviolet light absorption value of its 260nm and 280nm, calculate concentration and the purity of the DNA that extracts.According to the concentration of surveying dna solution is diluted to 100ng/ μ l and place-20 ℃ standby.
(2) integrity of detection genomic dna
With the nucleic acid mixed solution of genetically engineered soybean and transgenosis vegetable seed as template, use two endogenous reference genes (plant soybean lectin plain gene Lectin and phosphoric acid enol pyruvic acid carboxylase gene PEP) of soybean and vegetable seed to carry out pcr amplification simultaneously, confirm that mixed solution can be used as the pcr amplification template.The reaction system of PCR and reaction conditions are with embodiment 1 step (2), and difference only is that primer is Lectin-F, Lectin-R, PEP-F and PEP-R.
(3) GeXP multiple PCR technique
Following primer by waiting mole number to mix, is obtained primer C:
The upstream and downstream primer of foreign gene cauliflower mosaic virus 35S promoter (CaMV35S) is respectively:
CaMV35s-F?AGGTGACACTATAGAATAGCTCCTACAAATGCCATCA
CaMV35s-R：GTACGACTCACTATAGGGAGATAGTGGGATTGTGCGTCA；
The upstream and downstream primer of foreign gene 5-shikimic acid-3-phosphate synthase gene (EPSPS) is respectively:
EPSPS-F：AGGTGACACTATAGAATAGTTGCGGCCCTGCTTGTT
EPSPS-R：GTACGACTCACTATAGGGAGGCGGTCGCTTTCCTTGA；
The upstream and downstream primer of foreign gene rouge alkali synthetase gene terminator (NOS) is respectively:
NOS-F：AGGTGACACTATAGAATAATCGTTCAAACATTTGGCA
NOS-R：GTACGACTCACTATAGGGAATTGCGGGACTCTAATCATA；
The upstream and downstream primer of foreign gene bacillus grass fourth phosphinothricin acetyl transferase gene (BAR) is respectively:
Bar-F：AGGTGACACTATAGAATATCTGCACCATCGTCAACCACT
Bar-R：GTACGACTCACTATAGGGACTCCAGGGACTTCAGCAGGTG；
The upstream and downstream primer of foreign gene grass fourth phosphinothricin acetyl transferase gene (PAT) is respectively:
PAT-F：AGGTGACACTATAGAATACGGAGAGGAGACCAGTTGAG
PAT-R：GTACGACTCACTATAGGGATGGGTGTTTGTGGCTCTGT；
The upstream and downstream primer of foreign gene radix scrophulariae mosaic virus 35 S promoter (FMV35S) is respectively:
FMV35S-F：AGGTGACACTATAGAATAAAGACATCCACCGAAGACTTA
FMV35S-R：GTACGACTCACTATAGGGAAGGACAGCTCTTTTCCACGTT；
Following primer by waiting mole number to mix, is obtained primer D:
Upstream universal primer sequence: Cy5-AGGTGACACTATAGAATA
Downstream universal primer sequence: Cy5-GTACGACTCACTATAGGGA;
Get 200 μ L PCR pipe, add Ex Taq (Takara) 12.5 μ l, last, primer C (1 μ mol/L) 1.25 μ l, primer D (10 μ mol/L) 1.25 μ l, dna profiling 1 μ l, the sterilization distilled water complements to 25 μ l.The PCR reaction tubes is put into the PCR instrument, and amplification condition is 95 ℃ of 10min; 95 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 30s, 35 circulations; 72 ℃, 10min.
Extract reaction solution 1 μ l and carry out capillary electrophoresis:
The system of capillary electrophoresis is 40 μ l:
At this mixed solution of each Kong Zhongjun packing 39 μ l of last model, get the PCR product that 1 μ l step (3) obtains and add wherein behind 155: 1 by volume thorough mixings of ratio of SLS and DSS-400, blow and beat 3～4 times, cover a dropstone wax oil at last in every hole; Every hole adds dissociating buffer, carries out capillary electrophoresis;
The condition of capillary electrophoresis is:
Kapillary: 50 ℃ of temperature;
Sex change: 90 ℃, 120sec;
Inject sample: 2.0Kv, 30sec;
Separate: 6.0Kv, 35min;
This goes on foot used instrument: Beckman Genomelab Gexp-Genetic Analysis System.
The result can clearly observe 6 peaks as shown in Figure 3, judges gene according to X-coordinate size bp numerical values recited, wherein: NOS 202bp, CaMV35s 232bp, EPSPS 353bp, Bar 321bp, PAT 161bp, FMV35S 247bp.From left to right be respectively PAT, NOS, CaMV35S, FMV35S, Bar and EPSPS gene.As seen method provided by the present invention can detect transgene component exactly from soybean and vegetable seed mixture.
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a GeXP multiple PCR technique detects the method for transgene component in the plant, it is characterized in that may further comprise the steps:
(1) primer of design foreign gene and native gene, following primer is 5 '-3 ';
The upstream and downstream primer of foreign gene 5-shikimic acid-3-phosphate synthase gene is respectively:
EPSPS-F：AGGTGACACTATAGAATAGTTGCGGCCCTGCTTGTT
EPSPS-R：GTACGACTCACTATAGGGAGGCGGTCGCTTTCCTTGA；
The upstream and downstream primer of foreign gene cauliflower mosaic virus 35S promoter is respectively:
CaMV35s-F?AGGTGACACTATAGAATAGCTCCTACAAATGCCATCA
CaMV35s-R：GTACGACTCACTATAGGGAGATAGTGGGATTGTGCGTCA；
The upstream and downstream primer of foreign gene rouge alkali synthetase gene terminator is respectively:
NOS-F：AGGTGACACTATAGAATAATCGTTCAAACATTTGGCA
NOS-R：GTACGACTCACTATAGGGAATTGCGGGACTCTAATCATA；
The upstream and downstream primer of foreign gene bacillus grass fourth phosphinothricin acetyl transferase gene is respectively:
Bar-F：AGGTGACACTATAGAATATCTGCACCATCGTCAACCACT
Bar-R：GTACGACTCACTATAGGGACTCCAGGGACTTCAGCAGGTG；
The upstream and downstream primer of foreign gene grass fourth phosphinothricin acetyl transferase gene is respectively:
PAT-F：AGGTGACACTATAGAATACGGAGAGGAGACCAGTTGAG
PAT-R：GTACGACTCACTATAGGGATGGGTGTTTGTGGCTCTGT；
The upstream and downstream primer of foreign gene radix scrophulariae mosaic virus 35 S promoter is respectively:
FMV35S-F：AGGTGACACTATAGAATAAAGACATCCACCGAAGACTTA
FMV35S-R：GTACGACTCACTATAGGGAAGGACAGCTCTTTTCCACGTT；
The upstream and downstream primer of native gene phosphoric acid enol pyruvic acid carboxylase gene is respectively:
PEP-F：AGGTGACACTATAGAATAGCTAGTGTAGACCAGTTCTTG
PEP-R：GTACGACTCACTATAGGGACACTCTTGTCTCTTGTCCTC；
The upstream and downstream primer of native gene plant soybean lectin plain gene is respectively:
Lectin-F：AGGTGACACTATAGAATAGCCCTCTACTCCACCCCCATCC
Lectin-R：GTACGACTCACTATAGGGAGCCCATCTGCAAGCCTTTTTGTG；
The upstream universal primer is: AGGTGACACTATAGAATA; Wherein this primer carries out fluorescent mark;
The downstream universal primer is: GTACGACTCACTATAGGGA; Wherein this primer carries out fluorescent mark;
(2) genome of extraction sample;
(3) carry out multiplex PCR: the genome that obtains with step (2) is template, the primer of foreign gene and native gene described in the step (1) can independent assortment, described upstream universal primer and downstream universal primer are essential primer, carry out PCR, obtain the PCR product;
(4) capillary electrophoresis detects the PCR product;
(5) result is analyzed.
2. detect the method for transgene component in the plant according to the described GeXP multiple PCR technique of claim 1, it is characterized in that: the PCR described in the step (3) is the kind difference according to sample, selects primer:
1. when sample is soybean, primer be EPSPS-F, EPSPS-R, CaMV35s-F, CaMV35s-R, NOS-F, NOS-R, Lectin-F and Lectin-R by etc. the primer A that obtains of mixed in molar ratio, and upstream universal primer and downstream universal primer by etc. the primer D that obtains of mixed in molar ratio;
2. when sample is vegetable seed, primer be Bar-F, Bar-R, PAT-F, PAT-R, FMV35S-F, FMV35S-R, NOS-F, NOS-R, PEP-F and PEP-R by etc. the primer B that obtains of mixed in molar ratio, and upstream universal primer and downstream universal primer by etc. the primer D that obtains of mixed in molar ratio;
3. when sample is soybean and vegetable seed mixture, primer be EPSPS-F, EPSPS-R, CaMV35s-F, CaMV35s-R, Bar-F, Bar-R, PAT-F, PAT-R, FMV35S-F, FMV35S-R, NOS-F and NOS-R by etc. the primer C that obtains of mixed in molar ratio, and upstream universal primer and downstream universal primer by etc. the primer D that obtains of mixed in molar ratio.
3. detect the method for transgene component in the plant according to the described GeXP multiple PCR technique of claim 2, it is characterized in that: the reaction system of described PC R is: per 25 μ l PCR reaction systems contain: primer A, primer B or primer C 1.25pmol, primer D 12.5pmol, genome 20～500ng.
4. detect the method for transgene component in the plant according to the described GeXP multiple PCR technique of claim 2, it is characterized in that: the reaction conditions of described PC R is 95 ℃ of 10min; 95 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 30s, 35 circulations; 72 ℃ of 10min.
5. detect the method for transgene component in the plant according to the described GeXP multiple PCR technique of claim 1, it is characterized in that: described fluorescent mark carries out mark for using fluorescein Cy5.
6. detect the method for transgene component in the plant according to the described GeXP multiple PCR technique of claim 1, it is characterized in that: the operation steps of capillary electrophoresis is described in the step (4): use sample solution and 155: 1 by volume thorough mixings of DSS-400 on the sample, in each hole of last model, add the aforementioned mixed solution of 39 μ l then, getting the PCR product that 1 μ l step (3) obtains again adds wherein, piping and druming covers paraffin oil in every hole at last; Every hole adds dissociating buffer, carries out capillary electrophoresis.
7. detect the method for transgene component in the plant according to the described GeXP multiple PCR technique of claim 6, it is characterized in that: the add-on of described dissociating buffer is 250 μ l.
8. detect the method for transgene component in the plant according to the described GeXP multiple PCR technique of claim 1, it is characterized in that: the condition of capillary electrophoresis is described in the step (4):
Kapillary: 50 ℃ of temperature;
Sex change: 90 ℃, 120sec;
Inject sample: 2.0Kv, 30sec;
Separate: 6.0Kv, 35min.
9. each described GeXP multiple PCR technique of claim 1～8 detects the application of the method for transgene component in the plant, and it is characterized in that: described method is used for the detection of plant transgene component.
10. application according to claim 9 is characterized in that: described plant is soybean and/or vegetable seed.
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| CN103436595B (en) * | 2013-04-29 | 2015-07-08 | 冯家望 | LAMP detection primer group of NOS terminator, kit and detection method |
| CN104673908A (en) * | 2015-02-12 | 2015-06-03 | 暨南大学 | Method for detecting transgenic papaya through universal primer multiple PCR technology |
| CN105567833A (en) * | 2016-01-29 | 2016-05-11 | 江汉大学 | Detection method for soybean transgenic ingredients |
| CN105603079A (en) * | 2016-01-29 | 2016-05-25 | 江汉大学 | Testing method for transgenic ingredients in soybean oil |
| CN107805664B (en) * | 2016-09-07 | 2022-05-24 | 中国检验检疫科学研究院 | Composition, kit and method for identifying deer species by GeXP multiple PCR |
| CN112760413A (en) * | 2021-03-22 | 2021-05-07 | 苏州大学 | Application of public primer-mediated multiple quantitative PCR detection technology in transgenic soybean detection |
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