CN102517393B - Plasmid reference molecule of quantitative determination of nucleic acids from transgenic soybean GTS40-3-2 - Google Patents
Plasmid reference molecule of quantitative determination of nucleic acids from transgenic soybean GTS40-3-2 Download PDFInfo
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Abstract
The invention discloses a plasmid reference molecule which is suitable for the quantitative determination of nucleic acids from a transgenic soybean GTS40-3-2, comprising a structure-specific sequence and a strain-specific sequence of the transgenic soybean GTS40-3-2 and a specific fragment of a soybean endogenous reference gene Lectin. The invention is characterized by designing primers to obtain the structure-specific sequence and the strain-specific sequence of the transgenic soybean GTS40-3-2 and the specific fragment of the soybean endogenous reference gene Lectin by PCR amplification through the analysis of inserting the exogenous source of the transgenic soybean GTS40-3-2 in the gene sequence; constructing by molecular cloning to form a artificial recombinant plasmid molecule pXL02 in a plasmid molecule; detecting the phosphorus content of the plasmid reference molecule by inductively coupled plasma mass spectrometry (ICP-MS) to calculate the concentration of the plasmid reference molecule. According to the invention, the plasmid reference molecule constructed in the invention can completely replace a transgenic soybean GTS40-3-2 positive standard sample, and the plasmid reference molecule is completely suitable for the quantitative PCR analysis and detection of structure specificity and strain specificity of the transgenic soybean GTS40-3-2 sample.
Description
Technical field
The present invention relates to a kind of plasmid molecule of technical field of bioengineering, specifically, relate to a kind of turning because of plasmid control molecule and structure and quantivative approach for soybean GTS40-3-2 detection by quantitative.
Background technology
Genetically modified organism technology be known as " application the most rapidly biotechnology ", development along with biotechnology, increasing transgenic plant are applied to agriculture field, promote agriculture production, help increasing peasant income, for the food problem that solution Regularity Governing World Population Growth brings has been opened up new approach.So-called transgenic plant (Genetically modified organism, GMO) refer to and utilize biotechnology, the gene of separation or synthetic from different biologies is recombinated in vitro, then import recipient plant cell, make new gene in recipient cell, integrate, express, its regeneration plant can be by asexual or sexual breeding, foreign gene is entailed to offspring, and thus obtained genetically modified plant type is called transgenic plant.Take transgenic plant directly as food or the food produced as Raw material processing are called genetically modified food.Transgenic technology shifts gene between different plant species, transform biological genetic material, the target transition that it is wanted to necessary for human at aspects such as proterties, nutritional quality, consumption qualities, meet the mankind's various demands, for example, improve output, express the ability that special nutrition composition even obtains the antiviral or pest-resistant evil of antiweed.For example genetically engineered soybean is the genetically modified crops of whole world cultivated area maximum, genetically engineered soybean is mainly to have imported this genes encoding phosphoric acid enol form propanone acyl shikimic acid synthetic enzyme of Antiglyphosate gene EPSPS gene (5-enolpyruvylshikimate-3-phosphate synthase), make the injury that soybean can herbicide-tolerant glyphosate, greatly facilitate soybean farming production and administration.
But along with the extensive plantation of genetically modified crops in worldwide, its edible safety and environmental security have also caused consumers in general and national governments and associated mechanisms personnel's attention.Genetically modified crops produce in laboratory, are the metastatic genes of large span, great-jump-forward, are different from very long natural selection and the hybridization of close species.In theory, the generation of genetically modified crops the organic evolution speed of some species that has been artificial quickening.These species do not pass through the process of long-term natural selection, and the security of itself is unknown, and can exert an influence to existing diversity of organism.Therefore a lot of countries and regions are formulated genetically modified organism security control rules one after another, implement labeling system, and develop actively genetically modified crops detect Monitoring techniques.
Be applied to transgenic detection method and mainly contain two kinds: detection of nucleic acids, protein detection.Wherein nucleic acid detection method is because the reasons such as detection sensitivity is high, applied widely, easy and simple to handle have become the main detection methods of transgenic plant and products thereof.Nucleic acid is because content in biomass cells is relatively stable in addition, is also relatively not easy destroyedly in product processing, is therefore commonly used for the detection targets of transgenic plant and products thereof.
Fluorescent quantitative poly chain reaction (real-time Fluorescent Quantitative PCR, FQ-PCR) method is polymerase chain reaction (polymerase chain reaction, PCR) technology is a kind of, is the most frequently used quantitative detecting method of the current detection of nucleic acids of generally acknowledging.Polymerase chain reaction claims again outer-gene amplification technique, is a Protocols in Molecular Biology for the specific DNA fragmentation of external a large amount of amplifications at short notice.Nucleic acid quantification round pcr, especially real-time fluorescence quantitative PCR (real-time fluorescent quantitative PCR, the FQ-PCR) technology occurring in recent years, has realized the leap of PCR by qualitative to quantitative.Real-Time Fluorescent Quantitative PCR Technique adds the fluorophor of energy specific mark PCR product in PCR reaction system, utilizes the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, finally by typical curve, unknown template is carried out to quantitative analysis.Real-Time Fluorescent Quantitative PCR Technique is a kind of nucleic acid quantification and the analytical technology growing up on PCR Qualitative basis, this technology has been mixed together high sensitivity, the high specific of DNA hybridization technique and the quantitative advantage of the high precision of spectroscopic techniques of PCR, and easy and simple to handle, pollution is lacked, and has become the important tool in many researchs such as molecular biology and applied science.
In reality detects, reference material is very important.At present, the domestic reference material detecting for genetically modified organism also seldom, only has a kind of genetically engineered soybean powder reference material at present.Biometric unit leading in the world or laboratory have started to carry out the development of reference material for transgenosis quantitative PCR detection, for example be positioned at the subordinate's of Joint Research Centre of Belgian European Union reference material and measurement Research institute (IRMM), prepared and provide certified reference material (CRMs) the series standard product kind more than 60 that relates to 14 kinds of genetically modified crops, these standard substance are sold on market, but holding at high price of international certified reference material, can not meet the demand that increasing transgenosis detects.And, mostly existing these reference materials are the dried powder of the histoorgans such as the plant of original agricultural-food vegetable material or seed, in use also need therefrom to extract DNA, preparation, storage and the impact of mensuration process factors are numerous, be difficult to the amount that remains constant, the stability that is difficult to guarantee measurement, traceability is poor.
By contrast, plasmid molecule reference material has clear superiority aspect a lot, and plasmid molecule refers to a kind of linearizing recombinant plasmid molecule of the specific fragment that contains transgenosis testing goal foreign gene and internal standard gene.Empirical tests plasmid control molecule is good standard positive material surrogate in GMO identification and detection.The advantage of plasmid molecule is mainly to cultivate in a large number by microorganism, and DNA is easy to amplification, thus the reference material of unlimited stable quantity can be provided, and purity is higher; And processing ease, stability is high, and same plasmid molecule can comprise a plurality of external source goal gene simultaneously, and economy is efficient again.But imperfection is gone back in the development of plasmid molecule reference material at present, the positive criteria sample that a lot of testing laboratories are used lacks strict traceability and investigates, therefore detected result consistence, interoperability and reliability can not guarantee, in give detecting, quantitatively bring larger uncertainty, restricted the magnitude tracing development of China's transgenic product testing.
Summary of the invention
Technical problem to be solved by this invention is in order to overcome the difficult problem that in the nucleic acid quantification PCR detection of existing genetically engineered soybean GTS40-3-2, positive product lack and positive criteria product configure, and the plasmid control molecule of a kind of genetically engineered soybean GTS40-3-2 is provided.
The inventor is by inserting the analysis of gene order to the external source of genetically engineered soybean strain GTS 40-3-2-3-2, the amplification of design primer PCR obtains the specific fragment of its structure specific sequence, strain specificity sequence and soybean internal standard gene Lectin; Method by molecular cloning is building up to and in a plasmid molecule, forms artificial recombination plasmid molecule pXL02; The method of launching mass spectrum (ICP-MS) with inductively coupled plasma detects the phosphorus content of plasmid control molecule, thereby converses the concentration of plasmid control molecule.The plasmid control molecule building in the present invention can substitute genetically engineered soybean strain GTS 40-3-2-3-2 positive criteria product completely, this plasmid control molecule is applicable to completely to the structure specificity of genetically engineered soybean strain GTS 40-3-2-3-2 sample, strain specificity quantitative PCR analysis and detection, thoroughly solve the difficult problem that genetically engineered soybean strain GTS 40-3-2-3-2 detects Plays material want, and guaranteed the traceability of plasmid control molecule.
One of technical scheme that the present invention solves the problems of the technologies described above is: the plasmid control molecule of a kind of genetically engineered soybean GTS40-3-2, the specific fragment of the structure specific sequence that contains genetically engineered soybean GTS40-3-2, strain specificity sequence and soybean internal standard gene Lectin.
In the present invention, the structure specific sequence of described genetically engineered soybean GTS40-3-2, refer to one section of sequence of external source Insert Fragment in genetically engineered soybean GTS40-3-2 genome, this section of sequence can be used as the structure specific sequence of genetically engineered soybean GTS40-3-2, preferably, as the nucleotide fragments that sequence formed of the CTP4 sequence in its genome and CTP4 sequence both sides, its preferred form can be part 35S promoter sequence, CTP4 sequence and part CP4EPSPS sequence.Wherein, described " part " is the length of 50-500bp.There is this " part " sequence, can form the structure specific sequence of genetically engineered soybean GTS40-3-2.Its preferred form is the nucleotide fragments that the CP4 EPSPS sequence of 109bp long 35S promoter sequence, CTP4 sequence (long 216bp altogether) and 365bp forms.
The strain specificity sequence of described genetically engineered soybean GTS40-3-2, refer to one section of sequence in genetically engineered soybean GTS40-3-2 genome, this section of sequence can be used as the strain specificity sequence of genetically engineered soybean GTS40-3-2, preferably as the exogenous insertion vector sequence containing in its genome and the fragment that formed with the adjacent soybean gene group sequence of this exogenous insertion vector.The form of the strain specificity sequence preference of described genetically engineered soybean GTS40-3-2 can be part exogenous insertion vector sequence and the nucleotide fragments that forms with the adjacent part soybean gene group sequence of this exogenous insertion vector.Wherein, described " part " is the length of 50-500bp.There is this " part " sequence, can form the strain specificity sequence of genetically engineered soybean GTS40-3-2.The preferred form of strain specificity sequence of described genetically engineered soybean GTS40-3-2 is the soybean gene group sequence that comprises that the carrier sequence of 192bp and 127bp and this carrier sequence are adjacent.
In the present invention, described soybean internal standard gene Lectin, refers to soybean lectin plain gene, and it is conservative at soybean gene group camber, has the feature of non-specific in specificity between planting, kind and single copy number.The specific fragment of soybean internal standard gene Lectin described in the present invention, refers to the fragment in soybean lectin plain gene with soybean agglutinin gene specific.In soybean lectin plain gene, the fragment of the 1029bp～1579bp of full-length gene be have specific.The specific fragment of soybean internal standard gene Lectin of the present invention is exactly preferably the nucleotide fragments that is selected from the continuous 50-550bp of the 1029th to the 1579th of Lectin full-length gene, and optimum is exactly the fragment of the 1029th to the 1579th of Lectin full-length gene.
Two of technical scheme of the present invention is: the preparation method of the plasmid control molecule of a kind of described genetically engineered soybean GTS40-3-2, comprises the following steps:
1. utilize primer to pass through the specific fragment of structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of PCR specific amplification genetically engineered soybean GTS40-3-2;
2. the specific fragment of the structure specific sequence of the genetically engineered soybean GTS40-3-2 successively pcr amplification being obtained respectively, strain specificity sequence and soybean internal standard gene Lectin is cloned on cloned plasmids carrier, obtains plasmid control molecule pXL02.
In the present invention, the structure specific sequence of described genetically engineered soybean GTS40-3-2 is part 35S promoter sequence, CTP4 sequence and part CP4EPSPS sequence; The strain specificity sequence of described genetically engineered soybean GTS40-3-2 is the adjacent sequence of genetically engineered soybean GTS40-3-2 exogenous insertion vector and soybean gene group; Described soybean internal standard gene Lectin is soybean lectin plain gene.
In the present invention, described PCR primer is that length is the oligonucleotide chain of 28 ± 10nt, and it is identical or complementary with the specific fragment of structure specific sequence, strain specificity sequence or the soybean internal standard gene Lectin of genetically engineered soybean GTS40-3-2.
In the present invention, described specific amplification is the specific fragment that utilizes structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of the PCR primer specificity amplification genetically engineered soybean GTS40-3-2 designing.
In the present invention, described clone is that the specific fragment of structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of genetically engineered soybean GTS40-3-2 that above-mentioned pcr amplification is obtained is connected on plasmid vector successively according to the restriction enzyme digestion sites of design, obtains standard plasmid molecule pXL02.
In the present invention, described plasmid vector can be any conventional carrier, preferably cloning vector, the cloning vector that more preferably can breed in intestinal bacteria, as pUC19, pUC18,, pUC118, pUC119, pUC19, pBlueScript II SK or pGEM serial carrier.
Three of technical scheme of the present invention is: the quantivative approach of the plasmid control molecule of a kind of described genetically engineered soybean GTS40-3-2, comprises the following steps:
1. extract plasmid control molecule pXL02;
2. according to step 1. based composition and the sequence length of the plasmid control molecule pXL02 of gained, calculate the content of the phosphoric in plasmid control molecule pXL02 molecule;
3. prepare the phosphorus standardized solution of gradient concentration, and with this standardized solution, make the typical curve of inductively coupled plasma transmitting mass spectrum (ICP-MS).
4. ICP-MS detects plasmid control molecule pXL02 solution, and according to step 3. the typical curve of gained draw the phosphorus content of plasmid control molecule pXL02 solution.
5. according to the 4. 2. content of the phosphoric in the plasmid control molecule pXL02 molecule of gained of the phosphorus content of the plasmid control molecule pXL02 solution of gained and step of step, calculate the concentration of plasmid control molecule pXL02.
In the present invention, the preferred RF power of parameter of inductively coupled plasma transmitting mass spectrometric detection is 1100W, and atomization gas flow is 1.08L/min, and assisted gas flow is 1.2L/min, and isoelectronic species airshed is 16L/min.
Four of technical scheme of the present invention is: the nucleic acid quantification PCR detection method of a kind of genetically engineered soybean GTS40-3-2, institute's accepted standard material is described plasmid control molecule.
Beneficial effect of the present invention is, utilize the method for pcr amplification and molecular cloning to build the plasmid control molecule of the specific fragment of the structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin that contain genetically engineered soybean GTS40-3-2, and utilize inductively coupled plasma transmitting mass spectrum (ICP-MS) to carry out quantitatively, having guaranteed the traceability that it is good to it.Plasmid control molecule of the present invention can substitute the positive criteria material turning because of soybean GTS40-3-2, for the quantitative PCR detection of genetically engineered soybean GTS40-3-2 structure specificity, strain specificity.
Accompanying drawing explanation
Fig. 1 is the structure schema of the present invention one plasmid control molecule pXL02.
Fig. 2 is the collection of illustrative plates of the present invention one plasmid control molecule pXL02.
Embodiment
The invention provides plasmid control molecule and structure and quantivative approach that a kind of genetically engineered soybean GTS40-3-2 nucleic acid quantification detects use.This plasmid control molecule can substitute and turn because of soybean GTS40-3-2 positive criteria material, for the quantitative PCR detection of genetically engineered soybean GTS40-3-2 structure specificity, strain specificity.
The present invention, by the external source of genetically engineered soybean GTS40-3-2 being inserted to the analysis of gene order, designs PCR primer, obtains the specific fragment of its structure specific sequence, strain specificity sequence and soybean internal standard gene Lectin through pcr amplification; Method by molecular cloning is building up in a plasmid, thereby forms artificial recombination plasmid molecule pXL02; The method of launching mass spectrum (ICP-MS) with inductively coupled plasma detects the phosphorus content of plasmid positive criteria molecule, thereby converses the concentration of plasmid positive criteria molecule.
The plasmid control molecule that the present invention builds can substitute genetically engineered soybean GTS40-3-2 positive criteria material completely, this plasmid control molecule is applicable to completely to the structure specificity of genetically engineered soybean GTS40-3-2 sample, strain specificity quantitative PCR analysis and detection, thoroughly solve the difficult problem that in genetically engineered soybean GTS40-3-2 detection, positive reference material lacks, and guaranteed the traceability of plasmid control molecule.
Nucleic acid quantification PCR detects the principle of soybean GTS-40-3-2
Adopt real-time fluorescence PCR technology and can specific amplification genetically engineered soybean GTS-40-3-2 in structure gene or strain specificity gene and the primer of soybean Lectin gene and the probe of two ends mark fluorescent, amplification assay sample DNA, and Real-Time Monitoring PCR product respectively.Meanwhile, with the positive criteria material (or positive criteria molecule) of identical primer, probe and condition amplification concentration known, to obtain stable typical curve.According to the typical curve of foreign gene (structure specific gene or strain specific gene) and native gene, can calculate respectively the absolute content (copy number or concentration) of corresponding gene in sample, and calculate the relative content of genetically engineered soybean GTS-40-3-2 in test sample by absolute content.As adopt positive criteria to divide period of the day from 11 p.m. to 1 a.m calculating relative content should use gain factor.
Reference material
Reference material is to have one or more enough all even fine characteristic values of having determined, in order to correcting device, evaluates measuring method or to material or the material of material assignment.
Transgenic plant detection reference material comprises standard positive material and plasmid control molecule.
Standard positive material is the reference material that a class is made with primordial plant material, is the transgenic plant material isozygotying, and is used as the standard substance of positive control and configuration detection by quantitative, for GMO mixed-level is quantized and analyzed in PCR testing process.In transgenosis quantitative PCR detection process, positive criteria product are necessary, so the configuration requirement of positive criteria product is very strict, must can be applied to transgenosis quantitative PCR detection through the experimental verification in a plurality of laboratories.This class derives from the reference material of agricultural-food, and its preparation, storage and mensuration process are subject to a lot of Effects of Factors, is difficult to the amount that remains constant, and is not that all crop transgenic strains all exist standard positive material.
Plasmid control molecule
Plasmid control molecule refers to a kind of linearizing recombinant plasmid molecule of the specific fragment that contains transgenosis testing goal foreign gene and internal standard gene.Empirical tests plasmid control molecule is good standard positive material surrogate in GMO identification and detection.The advantage of plasmid molecule is mainly to cultivate in a large number by microorganism, and DNA is easy to amplification, thus the reference material of unlimited stable quantity can be provided, and purity is higher; And processing ease, stability is high, and same standard molecule can comprise a plurality of external source goal gene simultaneously, and economy is efficient again.Even there is scholar plasmid control molecule to be called to " gold standard material ".In transgenosis quantitative PCR detection process, standard molecule converts according to molecular size range and plant genome DNA molecule, thereby completes the quantitative analysis to transgenosis sample.Standard molecule can be applied to transgenosis PCR and detect, especially in quantitative PCR detection.
Plasmid control molecule of the present invention, the specific fragment of the structure specific sequence that contains genetically engineered soybean GTS40-3-2, strain specificity sequence and soybean internal standard gene Lectin.
In the present invention, the structure specific sequence of described genetically engineered soybean GTS40-3-2, refer to one section of sequence of external source Insert Fragment in genetically engineered soybean GTS40-3-2 genome, this section of sequence can be used as the structure specific sequence of genetically engineered soybean GTS40-3-2, preferably, as the nucleotide fragments that sequence formed of the CTP4 sequence in its genome and CTP4 sequence both sides, its preferred form can be part 35S promoter sequence, CTP4 sequence and part CP4 EPSPS sequence.Wherein, described " part " is the length of 50-500bp.There is this " part " sequence, can form the structure specific sequence of genetically engineered soybean GTS40-3-2.Its preferred form is the nucleotide fragments that the CP4 EPSPS sequence of 109bp long 35S promoter sequence, CTP4 sequence (long 216bp altogether) and 365bp forms.The structure specific sequence of described genetically engineered soybean GTS40-3-2 is preferably as shown in SEQ ID NO.1, and wherein the 1st to 109 is part 35S promoter sequence, and the 110th to 326 is CTP4 sequence, and the 327th to 691 is part CP4 EPSPS sequence.
The strain specificity sequence of genetically engineered soybean GTS40-3-2 of the present invention, refer to one section of sequence in genetically engineered soybean GTS40-3-2 genome, this section of sequence can be used as the strain specificity sequence of genetically engineered soybean GTS40-3-2, preferably as the exogenous insertion vector sequence containing in its genome and the fragment that formed with the adjacent soybean gene group sequence of this exogenous insertion vector.The form of the strain specificity sequence preference of described genetically engineered soybean GTS40-3-2 can be part exogenous insertion vector sequence and the nucleotide fragments that forms with the adjacent part soybean gene group sequence of this exogenous insertion vector.Wherein, described " part " can be the length of 20-500bp.There is this " part " sequence, can form the strain specificity sequence of genetically engineered soybean GTS40-3-2.The preferred form of strain specificity sequence of described genetically engineered soybean GTS40-3-2 is the soybean gene group sequence that comprises that the carrier sequence of 192bp and 127bp and this carrier sequence are adjacent.Preferably, the strain specificity sequence of described genetically engineered soybean GTS40-3-2 is as shown in SEQ ID NO.2, and wherein the 1st to the 192nd is exogenous insertion vector sequence, and wherein the 193rd to the 319th is adjacent soybean gene group sequence.
In the present invention, described soybean internal standard gene Lectin, refers to soybean lectin plain gene, and it is conservative at soybean gene group camber, has the feature of non-specific in specificity between planting, kind and single copy number.The specific fragment of soybean internal standard gene Lectin described in the present invention, refers to the fragment in soybean lectin plain gene with soybean agglutinin gene specific.In soybean lectin plain gene, the fragment of the 1029th～the 1579th of full-length gene be have specific.The specific fragment of soybean internal standard gene Lectin of the present invention is exactly preferably the nucleotide fragments that is selected from the continuous 50-550bp of the 1029th to the 1579th of Lectin full-length gene, and optimum is exactly the fragment of the 1029th to the 1579th of Lectin full-length gene.Preferably, the sequence of the specific fragment of described soybean internal standard gene Lectin is as shown in SEQ ID NO.3.
The sequence of plasmid control molecule pXL02 of the present invention is preferably as shown in SEQ ID NO.10, wherein the 1336th is structure specific sequence to the 2026th, the 986th to the 1305th is strain specificity sequence, and the 413rd is the specific fragment of internal standard gene Lectin to the 963rd.
Plasmid control molecule construction process
Plasmid control molecule pXL02 construction process of the present invention, comprises the following steps:
1. utilize database (such as Genbank, Shanghai City genetically modified organism security detect assessment sharing service platform http://www.shgmo.org/welcome.htm) to carry out bioinformatic analysis, obtain structure specific sequence, strain specificity sequence, the soybean internal standard gene Lectin sequence of genetically engineered soybean GTS40-3-2.
2. design PCR Auele Specific Primer;
Described PCR primer, refers to the oligonucleotide chain that length is 28 ± 10nt, and it is identical or complementary with the specific fragment of structure specific sequence, strain specificity sequence or the soybean internal standard gene Lectin of genetically engineered soybean GTS40-3-2.
Preferably, in the primer pair of the structure specific sequence of amplification genetically engineered soybean GTS40-3-2, the sequence of a primer is SEQ ID NO.4, and another is SEQ ID NO.5.
In the primer pair of the strain specificity sequence of amplification genetically engineered soybean GTS40-3-2, the sequence of a primer is SEQ ID NO.6, and another is SEQ ID NO.7.
In the primer pair of the specific fragment of the soybean internal standard gene Lectin of amplification genetically engineered soybean GTS40-3-2, the sequence of a primer is SEQ ID NO.8, and another is SEQ ID NO.9.
3. the specific fragment of the structure specific sequence of specific amplification genetically engineered soybean GTS40-3-2, strain specificity sequence and soybean internal standard gene Lectin.
So-called specific amplification, refers to the specific fragment of structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of the PCR primer specificity amplification genetically engineered soybean GTS40-3-2 that utilizes design.
4. the specific fragment of the structure specific sequence of the genetically engineered soybean GTS40-3-2 successively pcr amplification being obtained respectively, strain specificity sequence and soybean internal standard gene Lectin is cloned on cloned plasmids carrier, obtains plasmid control molecule pXL02;
Described clone, the specific fragment that refers to structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of the genetically engineered soybean GTS40-3-2 that above-mentioned pcr amplification is obtained is connected on plasmid vector successively according to the restriction enzyme digestion sites of design, obtains standard plasmid molecule pXL02.
Described plasmid vector can be any conventional carrier, preferably cloning vector, and the cloning vector that more preferably can breed in intestinal bacteria, as pUC19, pUC18,, pUC118, pUC119, pUC19, pBlueScript II SK or pGEM serial carrier.
5. the sequence of sequence verification plasmid control molecule pXL02.
6. the quantitative PCR detecting method of plasmid control molecule pXL02 checking.
Described quantitative PCR detecting method checking, refer to and detect the characteristics such as specificity, sensitivity, repeatability and repeatability of plasmid control molecule pXL02 when carrying out quantitative PCR analysis, to identify that this plasmid control molecule substitutes the ability of genetically engineered soybean GTS40-3-2 positive criteria material, and the mensuration validity that is applied to quantitative PCR detection genetically engineered soybean GTS40-3-2.
The quantivative approach of plasmid control molecule
The quantivative approach of plasmid control molecule pXL02 of the present invention, comprises the following steps:
1. extract plasmid control molecule pXL02;
2. according to the based composition of plasmid control molecule pXL02 and sequence length, calculate the content of the phosphoric of plasmid control molecule pXL02;
3. prepare the phosphorus standardized solution of gradient concentration, and with this standardized solution, make the typical curve of ICP-MS.
4. ICP-MS detects plasmid control molecule pXL02, and according to typical curve, draws the phosphorus content of plasmid control molecule pXL02.
5. according to the phosphorus content of plasmid control molecule pXL02, calculate the concentration of plasmid control molecule pXL02.
Below embodiments of the present invention are elaborated.The present embodiment is implemented under the prerequisite of technical solution of the present invention, has provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.In the following example, the experiment side of unreceipted actual conditions fails, conventionally according to normal condition, operate, as write molecular cloning with reference to Sambrook etc.: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) described condition, or the condition of advising according to manufacturer." room temperature " described in the present invention refers to the temperature of the operation room of testing, and is generally 25 ℃.
The structure of embodiment 1 plasmid control molecule
One, experiment reagent
Restriction enzyme KpnI, HindIII, XbaI, and the corresponding damping fluid of restriction enzyme is purchased from Shanghai Hao Jia development in science and technology company limited.
PUC19 carrier, Taq archaeal dna polymerase, T4DNA ligase enzyme, dNTP, DL2000 Marker
Purchased from Shanghai Hao Jia development in science and technology company limited;
DNA primer is synthetic by Shanghai Sheng Gong Bioisystech Co., Ltd.
Other biochemical reagents are import packing or domestic analytical pure.
Two, laboratory apparatus
DY-501 type nucleic acid electrophoresis apparatus (Shanghai Precision Scientific Apparatus Co., Ltd)
PTC-100 type pcr amplification instrument (MJ Research Inc.)
DNA electrophoretic analysis system, comprises camera bellows, digital camera, computer, scanner, ink-jet printer, Crystal shutter printer, Tanon UV-2000 uv analyzer, Gis gel image analysis software (Shanghai Tian Neng company)
Other Instruments comprises: whizzer, thermostat water bath, incubator, day equality.
Three, experimental technique and process
1, in GenBank, search for soybean internal standard gene Lectin; In Shanghai City genetically modified organism security, detect exogenous insertion vector that the upper inquiry of assessment sharing service platform (http://www.shgmo.org/welcome.htm) obtains genetically engineered soybean GTS40-3-2 concrete inserted mode and the copy number in genetically modified crops, and consult the sequence information of main element in exogenous insertion vector.
2, build the PCR primer sequence design of plasmid control molecule pXL02
Fig. 1 is shown in by the schematic diagram that builds plasmid control molecule pXL02.According to the genetically engineered soybean GTS40-3-2 external source insertion sequence information obtaining, utilize two pairs of PCR primers of software Primer 5.0 designs.Pair of primers is for the structure specific gene of the genetically engineered soybean GTS40-3-2 that increases, and a primer is positioned in 35S promoter sequence, and one is positioned in CP4 EPSPS gene order; Another pair of primers is for the strain specificity gene of the genetically engineered soybean GTS40-3-2 that increases, and a primer is positioned in soybean gene group, and another is positioned on the exogenous insertion vector of genetically engineered soybean GTS40-3-2.Design in addition pair of primers for the soybean internal standard gene Lectin specific sequence that increases.The two ends of each primer add restriction enzyme site and the protection base that molecular cloning is required.
In design of primers process, make every effort to make the plasmid that builds to be applicable to the detection of more genetically engineered soybean GTS40-3-2, the sequence comprising in plasmid both, applicable to the amplification of structure specific sequence, also applicable to strain specificity sequence amplification, will be taken into account the specificity of detected result simultaneously.
Concrete primer sequence is in Table 1.
Table 1. builds the PCR primer sequence of plasmid control molecule pXL02
3, the amplification of the structure specific sequence of genetically engineered soybean GTS40-3-2, strain specificity sequence and soybean internal standard gene order
According to the primer of table 1, the genetically engineered soybean GTS40-3-2 genomic dna of take is template, its structure specific sequence, strain specificity sequence and soybean internal standard gene order are carried out to pcr amplification (reaction system and condition are in Table 2,3), obtain the soybean internal standard Lectin gene order of the structure specific sequence of 709bp, the strain specificity sequence of 337bp and 570bp.Amplified production is reclaimed to purifying.
Table 2. builds the pcr amplification system of plasmid control molecule pXL02
| Reaction reagent | Consumption (μ L) |
| 10×buffer | 2 |
| dNTP(2.5mM) | 1 |
| Upstream primer (10 μ M) | 1 |
| Downstream primer (10 μ M) | 1 |
| Taq enzyme (2.5 units/reaction) | 1 |
| |
1 |
| ddH 2O | Complement to 20 μ L |
Table 3. builds the pcr amplification condition of plasmid control molecule pXL02
4, by PCR product cloning to plasmid vector pUC19
The structure specific sequence obtaining by restriction enzyme HindIII digest amplification respectively and carrier pUC19, reclaim structure specific sequence and linear plasmid pUC19 after enzyme is cut, T4 ligase enzyme connects, and connects product and transforms bacillus coli DH 5 alpha, plasmid molecule pUC19_1 in the middle of obtaining.The strain specificity sequence obtaining by restriction enzyme XbaI digest amplification respectively and middle plasmid molecule pUC19_1, interstitial granules pUC19_1 in strain specificity sequence after recovery enzyme is cut and linearity, T4 ligase enzyme connects, connect product and transform bacillus coli DH 5 alpha, plasmid molecule pUC19_2 in the middle of obtaining.The Lectin gene specific sequence obtaining by restriction enzyme KpnI digest amplification and middle plasmid molecule pUC19_2, interstitial granules pUC19_2 in Lectin sequence after recovery enzyme is cut and linearity, T4 ligase enzyme connects, connect product and transform bacillus coli DH 5 alpha, obtain plasmid control molecule pXL02, see Fig. 2.Endonuclease reaction system and linked system are in Table 4,5.
Table 4. endonuclease reaction system
| Reaction reagent | Consumption (μ L) |
| 10×buffer | 2 |
| |
1 |
| DNA | 7 |
| ddH 2O | Complement to 20 μ L |
Table 5. target DNA fragment ligation system
| Reaction reagent | Consumption (μ L) |
| 10 * T4 ligase enzyme buffer | 2 |
| T4 ligase enzyme | 2 |
| DNA fragmentation | 6 |
| Plasmid vector | 7 |
| ddH 2O | Complement to 20 μ L |
Experimental example 2 use ICP-MS carry out quantitatively the plasmid control molecule pXL02 building
One, experiment reagent
Phosphate radical-phosphorus composition analytical standard material 0.100g/L in GBW (E) 081216 water.
Two, laboratory apparatus
Inductively coupled plasma transmitting mass spectrograph (Perkin elmer ELAN DRC-e).
Three, experimental technique and process
1,, according to the based composition of plasmid control molecule pXL02 and sequence length, calculate the content of the phosphoric of plasmid control molecule pXL02;
2, ICP-MS experiment parameter is: RF power is 1100W, and atomization gas flow is 1.08L/min, and assisted gas flow is 1.2L/min, and isoelectronic species airshed is 16L/min.
3, the phosphorus standardized solution (0,10,50,100,200ppb) of preparation gradient concentration, and with this standardized solution, make the typical curve of ICP-MS;
4, ICP-MS detects plasmid control molecule pXL02, and according to typical curve, draws the phosphorus content of plasmid control molecule pXL02;
5, according to the phosphorus content of plasmid control molecule pXL02, calculate the concentration of plasmid control molecule pXL02;
Four, experimental result
According to the based composition of pXL02, calculate and learn that phosphorus content is wherein about 10.197%; According to the result of ICP-MS, the phosphorus content recording in the plasmid control molecule pXL02 of this batch of extraction is 15.957ppm, learns that the concentration of this crowd of plasmid control molecule pXL02 is about 3.63 * 10 after conversion
10copy/μ L.
The application of the plasmid control molecule pXL02 that embodiment 3 builds in reality detects
One, experiment reagent
Plant genome DNA extracts and purifying adopts the plant genome DNA of OMEGA company to extract test kit.
Extraction of plasmid DNA and purifying adopt the extraction of plasmid DNA test kit of OMEGA company.
PUC19 carrier, T4DNA ligase enzyme and damping fluid thereof, dNTPs, Taq archaeal dna polymerase and damping fluid thereof, DNA Marker are purchased from Shanghai Hao Jia development in science and technology company limited.
Primer and probe are synthetic by Shanghai Sheng Gong biotechnology company limited.
Other biochemical reagents are import packing or domestic analytical pure.
Two, laboratory apparatus
DY-501 type nucleic acid electrophoresis apparatus (Shanghai Precision Scientific Apparatus Co., Ltd).
Opticon-2 quantitative pcr amplification instrument (MJ Research Inc.).
DNA electrophoretic analysis system, comprises camera bellows, digital camera, computer, scanner, ink-jet printer, Crystal shutter printer, Tanon UV-2000 uv analyzer, Gis gel image analysis software (Shanghai Tian Neng company).
Other Instruments comprises: whizzer, thermostat water bath, incubator, day equality.
Three, experimental technique and process
1, DNA extraction and purifying
1) plant genome DNA extracts
A, get appropriate soybean sample, add liquid nitrogen grind into powder in mortar, take about 10-50mg powder to 1.5mL centrifuge tube, add 800 μ L Buffer P1;
B, 65 ℃ of incubations 10 minutes, mix twice therebetween;
C, add 140 μ L Buffer P2, vibration mixes.Centrifugal 10 minutes of >=10000 * g, carefully moves to supernatant in one new pipe;
D, add the Virahol of 0.7 times of volume, vibration is with precipitation DNA, and 10000 * g is centrifugal two minutes immediately;
E, supernatant discarded, add 300 μ L to be preheated to the sterilized water of 65 ℃, and vibration is with resuspended DNA.Add 4 μ L RNase A to mix;
F, add 150 μ L Buffer P3, then add 300 μ L dehydrated alcohols, vibration mixes;
G, all samples is joined in HiBind DNA adsorption column and (is placed in 2mL collection tube), centrifugal one minute of 10000 * g is with in conjunction with DNA.Discard collection tube and liquid wherein;
H, adsorption column is put into new collection tube, with 650 μ L DNA Wash Buffer (dehydrated alcohol dilution), rinse;
I, repeated washing step, rinse with 650 μ L DNA Wash Buffer (dehydrated alcohol dilution);
Centrifugal 2 minutes of j, maximum speed (being no more than 20000 * g) are to be dried adsorption column;
K, adsorption column is put into a clean 1.5mL pipe, add 100 μ L to be preheated to the Elution Buffer (or 10mM Tris buffer pH8.5/9.0 or aseptic deionized water) of 65 ℃, room temperature is placed 3-5 minute.Centrifugal 1 minute eluted dna of 10000 * g.
2) extraction of plasmid DNA
A, by the bacterium of 1.5-5.0mL incubated overnight in room temperature 10000 * g centrifugal 1 minute, supernatant discarded;
B, add 250 μ L Solution I (containing RNase A), vibration fully mixes;
C, add 250 μ L Solution II, the gentleness that turns upside down mixes, and room temperature is placed 2 minutes so that the abundant cracking of thalline;
D, add 350 μ L Solution III, fully put upside down up and down immediately and mix for several times, until form uniform white precipitate;
Centrifugal 10 minutes of e, >=13000 * g room temperature;
F, supernatant is moved in a clean HiBind adsorption column (being placed in 2mL collection tube) carefully to centrifugal 1 minute of 10000 * g room temperature;
G, discard the liquid in collection tube, add 500 μ L Buffer HB to clean adsorption columns, centrifugal 1 minute of 10000 * g room temperature;
H, discard the liquid in collection tube, add 700 μ L DNA Wash Buffer (dehydrated alcohol dilution) to clean adsorption columns;
I, repeat above-mentioned cleaning step;
Centrifugal 2 minutes of j, 13000 * g are to be dried adsorption column;
K, adsorption column is placed in to a clean 1.5mL pipe, adds 30 μ L-50 μ L Elution Buffer (or 10mM Tris buffer pH8.5) or sterilizing deionized waters, room temperature is placed 1-2 minute, and centrifugal 1 minute of 13000 * g is with eluted dna.
2, plasmid control molecule pXL02 is for limit of detection (LOD) and the quantitation limit (LOQ) of quantitative PCR detection
Employing standard GB/T 19495.5-2004 < < transgenic product detects PCR detection system and the reaction conditions in nucleic acid quantification PCR detection method > >, respectively with the plasmid control molecule pXL02 of different concns (as 3.63 * 10
6copies/ μ L, 3.63 * 10
5copies/ μ L, 3.63 * 10
4copies/ μ L, 3.63 * 10
3copies/ μ L, 3.63 * 10
2copies/ μ L, 3.63 * 10
1copies/ μ L, 3.63 * 10
0copies/ μ L) as standard substance, test LOD and the LOQ of 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, is determined while replacing positive criteria material with the plasmid control molecule pXL02 building the LOD of this quantitative PCR detecting method and LOQ.
3, repeatability and the repeatability of plasmid control molecule pXL02 detection by quantitative system
By standard GB/T 19495.5-2004 < < transgenic product, detect PCR detection system and the reaction conditions in nucleic acid quantification PCR detection method > >, respectively with the plasmid control molecule pXL02 of different concns (as 3.63 * 10
6copies/ μ L, 3.63 * 10
5copies/ μ L, 3.63 * 10
4copies/ μ L, 3.63 * 10
3copies/ μ L, 3.63 * 10
2copies/ μ L, 3.63 * 10
1copies/ μ L, 3.63 * 10
0copies/ μ L) carry out repeatability and repdocutbility test, each reacts in triplicate.According to the typical curve of quantitative pcr amplification, determine repeatability and the repeatability of the genetically engineered soybean GTS40-3-2 quantitative PCR reaction of setting up according to plasmid control molecule pXL02.
4, the quantitative analysis of application plasmid control molecule pXL02 to actual genetically engineered soybean GTS40-3-2 sample
By standard GB/T 19495.5-2004 < < transgenic product, detect PCR detection system and the reaction conditions in nucleic acid quantification PCR detection method > >, according to the quantitative PCR typical curve of drawing, the genetically engineered soybean GTS40-3-2 biased sample that is 10% to transgenosis content is analyzed, and determines whether the genetically engineered soybean GTS40-3-2 detection by quantitative plasmid control molecule building can be effectively applied to the detection by quantitative of actual genetically engineered soybean GTS40-3-2 sample.
Four, experimental result
1, plasmid control molecule pXL02 carries out limit of detection (LOD) and the quantitation limit (LOQ) of quantitative PCR detection
Employing standard GB/T 19495.5-2004 < < transgenic product detects PCR detection system and the reaction conditions in nucleic acid quantification PCR detection method > >, respectively with the plasmid control molecule pXL02 of different concns (as 3.63 * 10
6copies/ μ L, 3.63 * 10
5copies/ μ L, 3.63 * 10
4copies/ μ L, 3.63 * 10
3copies/ μ L, 3.63 * 10
2copies/ μ L, 3.63 * 10
1copies/ μ L, 3.63 * 10
0copies/ μ L) as standard substance, test LOD and the LOQ of quantitative PCR detecting method.Through 3 times, repeat experiment and determine that the LOD of this system is 3 copies, LOQ is 30 copies.Illustrate that the pXL02 plasmid control molecule building can replace genetically engineered soybean GTS40-3-2 positive criteria product to come detection by quantitative genetically engineered soybean GTS40-3-2 and converted products gm content thereof.
2, the repeatability of plasmid control molecule pXL02 detection by quantitative system and repeatability are measured
Employing standard GB/T 19495.5-2004 < < transgenic product detects PCR detection system and the reaction conditions in nucleic acid quantification PCR detection method > >, respectively with the plasmid control molecule pXL02 of different concns (as 3.63 * 10
6copies/ μ L, 3.63 * 10
5copies/ μ L, 3.63 * 10
4copies/ μ L, 3.63 * 10
3copies/ μ L, 3.63 * 10
2copies/ μ L, 3.63 * 10
1copies/ μ L, 3.63 * 10
0copies/ μ L) carry out repeatability and repdocutbility test, between 3 parallel reactors and 3 differences Ct value standard deviation of repeating to obtain between experiment be substantially all less than 0.2, repeatability and the repeatability of the genetically engineered soybean GTS40-3-2 quantitative PCR reaction that explanation is set up according to pXL02 plasmid control molecule are good, can be for the further quantitative analysis of actual genetically engineered soybean GTS40-3-2 sample.
3, the quantitative analysis of application plasmid control molecule pXL02 to actual genetically engineered soybean GTS40-3-2 sample
By standard GB/T 19495.5-2004 < < transgenic product, detect PCR detection system and the reaction conditions in nucleic acid quantification PCR detection method > >, according to the quantitative PCR typical curve of drawing, the genetically engineered soybean GTS40-3-2 biased sample that is 10% to transgenosis content is analyzed, quantitative analysis results shows that the transgenosis content of this sample is 11.54%, with the deviation of actual value be 15.4%, within the scope of the 0-0.25 that the deviation of detected result allows at ISO genetically modified food examination criteria, show that plasmid control molecule pXL02 that the present invention builds is applicable to quantitative PCR analysis and the detection of genetically engineered soybean GTS40-3-2.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. the plasmid control molecule of a genetically engineered soybean GTS40-3-2, it is characterized in that, the specific fragment of the structure specific sequence that contains genetically engineered soybean GTS40-3-2, strain specificity sequence and soybean internal standard gene Lectin, the structure specific sequence of described genetically engineered soybean GTS40-3-2 is as shown in SEQ ID NO.1, the strain specificity sequence of described genetically engineered soybean GTS40-3-2 is as shown in SEQ ID NO.2, and the sequence of the specific fragment of described soybean internal standard gene Lectin is as shown in SEQ ID NO.3.
2. the plasmid control molecule of genetically engineered soybean GTS40-3-2 as claimed in claim 1, is characterized in that, described plasmid control molecule is pXL02, and its sequence is as shown in SEQ ID NO.10.
3. a preparation method for the plasmid control molecule of genetically engineered soybean GTS40-3-2 as claimed in claim 1 or 2, is characterized in that, comprises the following steps:
1. utilize primer to pass through the structure specific sequence of PCR specific amplification genetically engineered soybean GTS40-3-2, the specific fragment of strain specificity sequence and soybean internal standard gene Lectin, wherein, in the primer pair of the structure specific sequence of amplification genetically engineered soybean GTS40-3-2, article one, the sequence of primer is SEQ ID NO.4, another is SEQ ID NO.5, in the primer pair of the strain specificity sequence of amplification genetically engineered soybean GTS40-3-2, article one, the sequence of primer is SEQ ID NO.6, another is SEQ ID NO.7, in the primer pair of the specific fragment of the soybean internal standard gene Lectin of amplification genetically engineered soybean GTS40-3-2, article one, the sequence of primer is SEQ ID NO.8, another is SEQ ID NO.9,
2. the specific fragment of the structure specific sequence of the genetically engineered soybean GTS40-3-2 successively pcr amplification being obtained respectively, strain specificity sequence and soybean internal standard gene Lectin is cloned on cloning vector, obtains plasmid control molecule pXL02.
4. the preparation method of the plasmid control molecule of genetically engineered soybean GTS40-3-2 as claimed in claim 3, it is characterized in that, described specific amplification is the specific fragment that utilizes structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of the PCR primer specificity amplification genetically engineered soybean GTS40-3-2 designing.
5. the preparation method of the plasmid control molecule of genetically engineered soybean GTS40-3-2 as claimed in claim 3, it is characterized in that, described clone is that the specific fragment of structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of genetically engineered soybean GTS40-3-2 that above-mentioned pcr amplification is obtained is connected on plasmid vector successively, obtains standard plasmid molecule pXL02.
6. a quantivative approach for the plasmid control molecule of genetically engineered soybean GTS40-3-2 as claimed in claim 1 or 2, is characterized in that, comprises the following steps:
1. extract plasmid control molecule pXL02;
2. according to step 1. based composition and the sequence length of the plasmid control molecule pXL02 of gained, calculate the content of the phosphoric in plasmid control molecule pXL02 molecule;
3. prepare the phosphorus standardized solution of gradient concentration, and with this standardized solution, make the typical curve of inductively coupled plasma transmitting mass spectrum (ICP-MS);
4. ICP-MS detects plasmid control molecule pXL02 solution, and according to step 3. the typical curve of gained draw the phosphorus content of plasmid control molecule pXL02 solution;
5. according to the 4. 2. content of the phosphoric in the plasmid control molecule pXL02 molecule of gained of the phosphorus content of the plasmid control molecule pXL02 solution of gained and step of step, calculate the concentration of plasmid control molecule pXL02.
7. the quantivative approach of the plasmid control molecule of genetically engineered soybean GTS40-3-2 as claimed in claim 6, it is characterized in that, ICP-MS parameter is as follows: RF power is 1100W, and atomization gas flow is 1.08L/min, assisted gas flow is 1.2L/min, and isoelectronic species airshed is 16L/min.
8. a nucleic acid quantification PCR detection method of genetically engineered soybean GTS40-3-2, is characterized in that, institute's accepted standard material is plasmid control molecule as claimed in claim 1 or 2.
9. the application of plasmid control molecule as claimed in claim 1 or 2 in the nucleic acid quantification PCR of genetically engineered soybean GTS40-3-2 detects.
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| CN103484557A (en) * | 2013-10-15 | 2014-01-01 | 上海市计量测试技术研究院 | Plasmid standard molecule applicable to real-time fluorescence quantification PCR (Polymerase Chain Reaction) detection of enterobacter sakazakii |
| CN103497964A (en) * | 2013-10-15 | 2014-01-08 | 上海市计量测试技术研究院 | Plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection |
| CN103497962A (en) * | 2013-10-15 | 2014-01-08 | 上海市计量测试技术研究院 | Plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection |
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| CN111218520A (en) * | 2019-10-26 | 2020-06-02 | 大连海关技术中心 | Transgenic soybean GTS-40-3-2 strain EFIRM detection probe and application thereof |
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| CN1769488A (en) * | 2005-10-21 | 2006-05-10 | 天津师范大学 | Transgenic soybean detection method and the primer |
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