CN112251538B - Method for analyzing herbicide-resistant transgenic soybean J12 copy number by microdroplet digital PCR (polymerase chain reaction) - Google Patents
Method for analyzing herbicide-resistant transgenic soybean J12 copy number by microdroplet digital PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention discloses a method for analyzing the copy number of herbicide-resistant transgenic soybean J12 by microdroplet digital PCR (polymerase chain reaction), which uses the herbicide-resistant transgenic soybean J12G2‑epspsAndGATexogenous insertion of target gene sequence, design and synthesis of specific primer and probe of 3' end transformant specific sequence, and combination with soybean internal standard geneLectinAnd constructing a microdroplet digital PCR reaction system. Droplet digital PCR (ddPCR) is a new absolute quantitative technology which is started in recent years, theoretical single-molecule amplification is realized through extreme dilution, then the original concentration of a sample is calculated by using end-point PCR and Poisson distribution, a standard product is not needed, a standard curve is not needed to be constructed, and the copy number of the sample is calculated through the ratio of the copy number concentration of an exogenous gene and an internal standard gene. The microdroplet digital PCR method is an economic, rapid and accurate new method for analyzing the copy number of the exogenous gene, has high sensitivity and accuracy, and can be widely applied to copy number analysis.
Description
Technical Field
The invention belongs to the technical field of agricultural transgenic organism safety evaluation, and particularly relates to a construction method and application of a method for analyzing the copy number of a new herbicide-resistant gene soybean variety based on a microdroplet digital PCR technology.
Background
With the development of transgenic technology, the safety problem of transgenic crops and derived foods thereof is strongly concerned by various countries, organizations and regions in the world, and clear policies and regulations are stipulated for the marketing and popularization of the transgenic crops, so that strict biosafety evaluation is carried out on the transgenic crops. Molecular characteristics such as expression vectors, target gene integration conditions, exogenous insertion sequence expression conditions and the like are important contents for safety evaluation of transgenic organisms, wherein copy number identification of exogenous genes inserted into transgenic crops is a key parameter. The position and copy number of the foreign gene integrated into the receptor genome affect the expression and genetic stability of the target gene, while the copy number of the inserted gene is more important than the insertion site. When a foreign gene is integrated into a receptor genome at a low copy number (1-2), high-efficiency transcription expression can be stabilized, and the integration at multiple copy numbers can cause unstable expression and even silence of the gene. Therefore, it is very important to identify the number of inserted copies of the foreign gene in the transgenic product.
At present, southern blot and real-time fluorescent quantitative PCR are two commonly used exogenous gene copy number analysis techniques, and have been widely used for exogenous gene copy number analysis. However, the two methods also have certain defects, and the Southern blot analysis method has large workload, long period and high operation requirement, and particularly has small result when being used for the analysis of multi-copy genes; the q RT-PCR method requires a standard curve to be constructed in analysis, the quantification by using the standard curve is a relative quantification method which is not very accurate, and the quality of the standard curve is easily influenced by various factors such as DNA purity, the concentration of primers and probes, reaction inhibition factors and the like, so the q RT-PCR method is not an ideal exogenous gene copy number analysis method. In recent years, droplet-based digital PCR (dd PCR) is a recently emerging nucleic acid detection technique. The technology does not depend on any calibrator, does not need to construct a standard curve, dilutes the nucleic acid by thousands of times in a form of forming a large number of water-in-oil droplets, then carries out PCR reaction by taking each small droplet as an independent reaction unit, and finally realizes absolute quantification of nucleic acid detection by utilizing the Poisson distribution principle. Compared with other methods, the method has the advantages of small workload, short period, low operation requirement, high accuracy and good repeatability, and becomes a preferred method for detecting the copy number of the foreign gene.
The herbicide-resistant transgenic soybean J12 is developed by the research institute of crop science of Chinese academy of agricultural sciencesG2-EPSPSGenes andGATa new herbicide-resistant soybean strain. The researchers adopt the agrobacterium-mediated transformation method to transformG2-EPSPSAndGATDNA introduction into the expression vector of two genesIn a soybean receptor, a J12 transformant with resistance to glyphosate is obtained through multi-generation screening, a new material with transgenic soybean independent intellectual property rights is created aiming at the common and serious wasteland of soybean production in China, scientific and technological support is provided for improving the competitiveness of the transgenic soybean industry in China, and the transgenic soybean transgenic plant has important industrial application prospects in China. The study is carried out with J12G2-epspsAndGATthe target gene sequence is inserted from the external source, the specific sequence of the transformant at the 3' end is taken as a target, andLectina micro-drop digital PCR copy number analysis method is established for internal standard genes through primer probe screening, specificity testing and the like, and aims to provide important technical support for safety evaluation, administrative supervision and intellectual property protection of the transformant.
Disclosure of Invention
The invention overcomes the defects of large workload, long period, high operation requirement and the like of two commonly used exogenous gene copy number analysis technologies of Southern blot and real-time fluorescent quantitative PCR, and provides a method for quickly, simply and accurately analyzing the insertion copy number of the exogenous target gene of the new transgenic soybean strain J12 in a genome. The invention aims to establish a microdroplet digital PCR copy number analysis method for a new transgenic soybean strain J12, and compared with other methods, the method has the advantages of small workload, short period, low operation requirement, high accuracy, good repeatability and the like.
The technical content of the invention is as follows:
a primer and a probe for analyzing the J12 copy number of herbicide-resistant transgenic soybeans are characterized by comprisingG2- epspsExogenous insertion of the target gene sequence:
forward primer sequence: 5'-TCGAGATTGATGGTGGTTTGTC-3'
Reverse primer sequence: 5'-TCAGCGCCACTTCAATCG-3'
The probe sequence is as follows: FAM-GGGCAAACTGATTTCCATAGCTT-BHQ1;
GATexogenous insertion of the target gene sequence:
forward primer sequence: 5'-GGGCAAACTGATTTCCATAGCTT-3'
Reverse primer sequence: 5'-CCTCGGAGCTGGTACTGTTTCT-3'
The probe sequence is as follows: FAM-ATTCCACCAGGCCGAGCACTCAGA-BHQ1;
3' end transformant specific sequence:
forward primer sequence: 5'-GCAGCTTGAGCTTGGATCAGA-3'
Reverse primer sequence: 5'-TCATAGCGTGGTGTTTGACATAAA-3'
The probe sequence is as follows: FAM-TGTCGTTTCCCGCCTTCAGTTTAAACC-BHQ1;
endogenous geneLectinThe gene sequence is as follows:
forward primer sequence: 5'-GCCCTCTACTCCACCCCCA-3'
Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;
the invention further discloses a method for analyzing the copy number of herbicide-resistant transgenic soybean J12, which is characterized by comprising the following steps:
（1）G2-epspsexogenous insertion of the target gene sequence:
forward primer sequence: 5'-TCGAGATTGATGGTGGTTTGTC-3'
Reverse primer sequence: 5'-TCAGCGCCACTTCAATCG-3'
The probe sequence is as follows: FAM-GGGCAAACTGATTTCCATAGCTT-BHQ1;
GATexogenous insertion of the target gene sequence:
forward primer sequence: 5'-GGGCAAACTGATTTCCATAGCTT-3'
Reverse primer sequence: 5'-CCTCGGAGCTGGTACTGTTTCT-3'
The probe sequence is as follows: FAM-ATTCCACCAGGCCGAGCACTCAGA-BHQ1;
3' end transformant specific sequence:
forward primer sequence: 5'-GCAGCTTGAGCTTGGATCAGA-3'
Reverse primer sequence: 5'-TCATAGCGTGGTGTTTGACATAAA-3'
The probe sequence is as follows: FAM-TGTCGTTTCCCGCCTTCAGTTTAAACC-BHQ1;
endogenous geneLectinThe gene sequence is as follows:
forward primer sequence: 5'-GCCCTCTACTCCACCCCCA-3'
Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;
(2) And (3) PCR reaction system: the total volume is 20 mu L, the method comprises 10 mu L of BIO-RAD ddPCR Supermix for Probes, 1 mu L of 10 mu mol/L upstream primer, 1 mu L of 10 mu mol/L downstream primer, 1 mu L of 10 mu mol/L probe and 1 mu L of 25 ng/mu L DNA template, and the final volume is supplemented to 20 mu L by sterile water;
(3) Droplet generation: carefully transferring a 20 mu L reaction system to a micro-card generator to avoid generating bubbles, simultaneously adding 70 mu L of microdroplet generation oil at a position corresponding to the micro-card generator, covering a gasket, putting the microdroplet generation oil into the microdroplet generator to generate microdroplets, tilting a pipettor at 30-45 degrees to suck 40 mu L of reaction products after the microdroplets are generated, carefully transferring the reaction products to a digital 96-well reaction plate, and heat-sealing the film at 170 ℃;
(4) PCR reaction procedure: 94. pre-variable thermal activation at 10 min for 1 cycle; 94. denaturation at 30 s, annealing at 60 deg.C for 1 min, and 40 cycles; enzyme inactivation at 98 deg.C for 10 min,1 cycle;
(5) And (4) analyzing results: automatically calculating the copy number concentration, the total microdroplet number and the positive microdroplet number of each reaction by a microdroplet digital PCR instrument, taking the total microdroplet number more than 10000 as an effective result, and calculating the copy number of the exogenous target gene of the herbicide-resistant transgenic soybean J12 in the sample inserted into the genome according to the formula A = B/C; in the formula, A is the copy number of the exogenous target gene of the herbicide-resistant transgenic soybean J12 inserted into the genome, B is the copy number concentration of the exogenous gene sequence of the herbicide-resistant transgenic soybean J12, and C is an internal reference geneLectinCopy number concentration of the gene.
The invention further discloses application of specific primers and probes for analyzing the copy number of the transgenic soybean J12 in rapid and efficient analysis of the copy number of the transgenic soybean J12 by using a microdroplet digital PCR technology. Experimental results show that the method greatly improves the working efficiency, has the advantages of rapidness, flexibility, high efficiency, small required sample amount and the like, can be used for quickly, conveniently and efficiently analyzing the copy number of the transgenic soybean J12 strain, and can provide reference for establishing a micro-drop digital PCR copy number analysis method of other transgenic soybean strains and other transgenic crops.
The invention mainly considers the specificity of a primer probe for analyzing the copy number of the transgenic soybean J12 by a microdroplet digital PCR technology and the amplification effect of microdroplets, simultaneously verifies the homozygosity of the new transgenic soybean J12 strain by using the copy number of a specific sequence of a 3' end transformant, and mainly solves the establishment of a method for analyzing the copy number of the transgenic soybean J12 by the microdroplet digital PCR technology.
The test effect of the invention is as follows:
(1) Using genome DNA of transgenic corn mixed sample, transgenic soybean mixed sample, transgenic rape mixed sample, transgenic rice mixed sample, transgenic cotton mixed sample and non-transgenic soybean as templateG2-epspsAndGATtarget gene primers are inserted from exogenous sources, and transformant specific primers at the 3' end are used for carrying out microdroplet digital PCR amplification. The results are shown in FIGS. 1-3, and positive droplets are generated only when herbicide-resistant transgenic soybean J12 genome DNA is used as a template, while no positive droplets are generated when other transgenic crops and non-transgenic soybean genome DNA are used as templates, which indicates that the specificity of the detection method is good.
(2) Using the genome DNA of herbicide-resistant transgenic soybean J12 as a template and the standard gene in the soybeanLectinFor reference genes, microdroplet digital PCR analysis was performed using the constructed microdroplet digital PCR detection system to determine the copy number concentration of each gene. The results are shown in FIGS. 4-6, internal standards for SoybeanLectinGenes andG2-epspsthe copy number concentration of the exogenously inserted target gene is 2780 copies/. Mu.L and 2620 copies/. Mu.L respectively, the soybean internal standardLectinGenes andGATthe copy number concentration of the exogenously inserted target gene is 1370 copies/. Mu.L and 1340 copies/. Mu.L respectively, the soybean internal standardLectinThe copy number concentration of the gene and the transformant-specific sequence at the 3' end were 955copies/. Mu.L and 909 copies/. Mu.L.
(3) Calculating the J12 copy number of the herbicide-resistant transgenic soybean in the sample
The copy number concentration and the endogenous gene of the herbicide-resistant transgenic soybean J12 target gene and the transformant specific sequenceLectinThe copy number concentration is substituted into a formula, and the copy number of the exogenous target gene of the herbicide-resistant transgenic soybean J12 in the sample, which is inserted into the genome, is calculated. Formula for calculating the herbicide-resistant transgenic soybean J12 copy number in the sample:
a-copy number of the exogenous target gene of herbicide-resistant transgenic soybean J12 in the sample inserted into the genome;
b-exogenous target gene copy number concentration of herbicide-resistant transgenic soybean J12;
C—Lectinendogenous gene copy number concentration;
and (4) test conclusion:
the research establishes a method for analyzing the copy number of the transgenic soybean J12 by a droplet type digital PCR. The method uses herbicide-resistant transgenic soybean J12 new lineG2-epspsAndGATthe target gene sequence is inserted into the exogenous gene sequence, the specific sequence of the 3' end transformant is the target sequence, PCR amplification primers and TaqMan probes are designed, the specificity of the primers and the probes is identified, and the soybean internal standard is used as the standardLectinThe gene is used as reference, and a micro-drop digital PCR copy number detection system is established. The specific experiment result shows that only the herbicide-resistant transgenic soybean J12 genome DNA is used as a template to have an amplification signal; exogenous target gene is carried out by using single transgenic soybean J12 genome DNAG2-epspsAndGATmicro-droplet digital PCR amplification of 3' end transformant specific sequence, exogenous target gene of transgenic soybean J12G2-epspsAndGATthe copy number of the insert on the genome is 0.94 and 0.98 respectively, and the copy number of the specific sequence of the 3' end transformant is 0.95, so that the single transgenic soybean J12 is verified to be homozygote, and the single transgenic soybean J12 is identifiedIs inserted as a single copy. The result shows that the transgenic soybean J12 copy number analysis method established by the research has the advantages of small workload, short period, low operation requirement, high accuracy and good repeatability, can be quickly, conveniently and efficiently used for analyzing the copy number of the new transgenic soybean J12 strain, can verify the homozygosity of the transgenic soybean material, and can provide reference for establishing the micro-drop digital PCR copy number analysis methods of other transgenic soybean strains and other transgenic crops.
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FIG. 1: exogenous target geneG2-epspsDroplet digital PCR specificity detection result; as noted in the figure: 1-2, blank control; 3-4, transgenic herbicide tolerant soybean J12;5-6, non-transgenic soybean samples; 7-8, mixing transgenic corn samples; 9-10, mixing other transgenic soybeans; 11-12, mixing the transgenic rape; 13-14, mixing the transgenic rice; 15-16, transgenic cotton mixed sample;
FIG. 2: exogenous target geneGATDroplet digital PCR specificity detection result; as the figure marks: 1-2, blank control; 3-4, transgenic herbicide tolerant soybean J12;5-6, non-transgenic soybean samples; 7-8, mixing transgenic corn samples; 9-10, mixing other transgenic soybeans; 11-12, mixing transgenic rape; 13-14, mixing transgenic rice samples; 15-16, transgenic cotton mixed sample;
FIG. 3: microdroplet digital PCR (polymerase chain reaction) specificity detection results of the transformant specificity sequences at the 3' end; as noted in the figure: 1-2, blank control; 3-4, transgenic herbicide tolerant soybean J12;5-6, non-transgenic soybean samples; 7-8, mixing transgenic corn samples; 9-10, mixing other transgenic soybeans; 11-12, mixing transgenic rape; 13-14, mixing the transgenic rice; 15-16, transgenic cotton mixed sample;
FIG. 4: microdroplet digital PCR amplification results of exogenous target genes G2-epsps and endogenous Lectin genes; as the figure marks: 1-2, endogenous Lectin gene copy number concentration; 3-4, copy number concentration of exogenous target gene G2-epsps; (upper panel for droplet amplification and lower panel for copy number concentration);
FIG. 5: microdroplet digital PCR amplification results of exogenous target genes GAT and endogenous Lectin genes; as noted in the figure: 1-2, copy number concentration of endogenous Lectin gene; 3-4, exogenous gene of interest GAT copy number concentration (upper panel for microdroplet amplification, lower panel for copy number concentration);
FIG. 6: microdroplet digital PCR 3' end transformant specific sequence and endogenous Lectin gene amplification result; as noted in the figure: 1-2, endogenous Lectin gene copy number concentration; 3-4,3' end transformant specific sequence copy number concentration (upper panel for microdroplet amplification, lower panel for copy number concentration).
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications can be made in the components and amounts of the materials used in these embodiments without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are commercially available, and the herbicide-resistant transgenic soybean J12 is researched and developed by the crop scientific research institute of Chinese academy of agricultural sciences and can be freely provided for research and use in scientific research.
Example 1
(1) Sample 1: transgenic herbicide-resistant soybean J12 random individual samples.
(2) Reagent: BIO-RAD ddPCR Supermix for Probes premix; endogenous source of soybean synthesized in ShanghaiLectinGene, exogenous target gene and transformant specific sequence amplification primer and probe.
G2-epspsExogenous insertion of a target gene sequence:
forward primer sequence: 5'-TCGAGATTGATGGTGGTTTGTC-3'
Reverse primer sequence: 5'-TCAGCGCCACTTCAATCG-3'
The probe sequence is as follows: FAM-GGGCAAACTGATTTCCATAGCTT-BHQ1;
GATexogenous insertion of the target gene sequence:
forward primer sequence: 5'-GGGCAAACTGATTTCCATAGCTT-3'
Reverse primer sequence: 5'-CCTCGGAGCTGGTACTGTTTCT-3'
The probe sequence is as follows: FAM-ATTCCACCAGGCCGAGCACTCAGA-BHQ1;
3' end transformant specific sequence:
forward primer sequence: 5'-GCAGCTTGAGCTTGGATCAGA-3'
Reverse primer sequence: 5'-TCATAGCGTGGTGTTTGACATAAA-3'
The probe sequence is as follows: FAM-TGTCGTTTCCCGCCTTCAGTTTAAACC-BHQ1;
endogenous geneLectinThe gene sequence is as follows:
forward primer sequence: 5'-GCCCTCTACTCCACCCCCA-3'
Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;
(3) And (3) PCR reaction system: the total volume is 20 mu L, and the kit comprises 10 mu L of BIO-RAD ddPCR Supermix for Probes, 1 mu L of upstream primer (10 mu mol/L), 1 mu L of downstream primer (10 mu mol/L), 1 mu L of probe (10 mu mol/L), 1 mu L of DNA template (25 ng/mu L), and finally the total volume is supplemented to 20 mu L by sterile water.
(4) Droplet generation: carefully transferring a 20 mu L reaction system to a micro-card generator to avoid generating bubbles, simultaneously adding 70 mu L of microdroplet generation oil at a position corresponding to the micro-card generator, covering a gasket, putting the microdroplet generation oil into the microdroplet generator to generate microdroplets, tilting a pipettor at 30-45 degrees to suck 40 mu L of reaction products after the microdroplets are generated, carefully transferring the reaction products to a digital 96-well reaction plate, and heat-sealing the film at 170 ℃;
(5) PCR reaction procedure: 94. pre-variable thermal activation at 10 min for 1 cycle; 94. denaturation at 30 s, annealing at 60 deg.C for 1 min, and 40 cycles; enzyme inactivation at 98 deg.C for 10 min,1 cycle.
(6) And automatically calculating the copy number concentration, the total microdroplet number and the positive microdroplet number of each reaction by a microdroplet digital PCR instrument, wherein the effective result is regarded as that the total microdroplet number is more than 10000. According to the formula A = B/C, the insertion of the exogenous target gene of the herbicide-resistant transgenic soybean J12 in the sample on the genome is calculatedThe number of copies to be copied. Copy number concentration of herbicide-resistant transgenic soybean J12 exogenous gene sequence and endogenous geneLectinThe copy number concentration of (a) is substituted into the formula.
The results are shown in table 1, the copy numbers of the exogenous target gene of the herbicide-resistant transgenic soybean J12 in the sample are calculated to be 0.94 and 0.98 respectively, the single-copy insertion is identified, and the single-plant soybean is identified as a homozygote through the copy number of the transformant specific sequence.
In the formula:
a-copy number of the exogenous target gene of herbicide-resistant transgenic soybean J12 in the sample inserted into the genome;
b-copy number concentration of the herbicide-resistant transgenic soybean J12 exogenous gene sequence;
C—Lectincopy number concentration of the endogenous gene;
TABLE 1 random Individual plant sample test results
And (4) test conclusion:
the method for analyzing the copy number of the transgenic soybean J12 by using the micro-drop digital PCR established by the research uses the genome DNA of the single transgenic soybean J12 to carry out exogenous target geneG2-epspsAndGATmicro-droplet digital PCR amplification of 3' end transformant specific sequence, exogenous target gene of transgenic soybean J12G2-epspsAndGATthe copy number of the insert on the genome is 0.94 and 0.98 respectively, and the copy number of the transformant specific sequence at the 3' end is 0.95, so that the single transgenic soybean J12 is verified to be homozygote, and the single transgenic soybean J12 is identified to be a single copy insert. The result shows that the transgenic soybean J12 copy number analysis method established by the research has the advantages of small workload, short period, low operation requirement, high accuracy and good repeatability, and can be quickly, conveniently and efficiently used for the transgenic soybeanDue to the copy number analysis of the new soybean J12 strain, the homozygosity of the transgenic soybean material is verified, and a reference can be provided for the establishment of a micro-drop digital PCR copy number analysis method of other transgenic soybean strains and other transgenic crops.
It will be apparent to those skilled in the art that various changes and modifications can be made in the above embodiments without departing from the scope and spirit of the invention, and it is intended that all such changes and modifications as fall within the true spirit and scope of the invention be interpreted in accordance with the principles of the invention. And the invention is not limited to the example embodiments set forth in the description.
SEQUENCE LISTING
<110> Tianjin City academy of agricultural sciences
<120> method for analyzing J12 copy number of herbicide-resistant transgenic soybean by microdroplet digital PCR (polymerase chain reaction)
<160> 12
<170> PatentIn version 3.5
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Claims (1)
1. Application of primer and probe for analyzing copy number of herbicide-resistant transgenic soybean J12 in identification of soybean J12 is characterized by comprising detectionG2-epspsPrimers and probes for exogenously inserting target gene sequences:
forward primer sequence: 5'-TCGAGATTGATGGTGGTTTGTC-3'
Reverse primer sequence: 5'-TCAGCGCCACTTCAATCG-3'
The probe sequence is as follows: FAM-GGGCAAACTGATTTCCATAGCTT-BHQ1;
detecting GATPrimers and probes for exogenously inserting target gene sequences:
forward primer sequence: 5'-GGGCAAACTGATTTCCATAGCTT-3'
Reverse primer sequence: 5'-CCTCGGAGCTGGTACTGTTTCT-3'
The probe sequence is as follows: FAM-ATTCCACCAGGCCGAGCACTCAGA-BHQ1;
detection of3' endPrimers and probes for transformant-specific sequences:
forward primer sequence: 5'-GCAGCTTGAGCTTGGATCAGA-3'
Reverse primer sequence: 5'-TCATAGCGTGGTGTTTGACATAAA-3'
The probe sequence is as follows: FAM-TGTCGTTTCCCGCCTTCAGTTTAAACC-BHQ1;
detection of endogenous genesLectinPrimers and probes for gene sequences:
forward primer sequence: 5'-GCCCTCTACTCCACCCCCA-3'
Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1.
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