CN102517393A - 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 commentaries on classics because of soybean GTS40-3-2 detection by quantitative with plasmid control molecule and structure and quantivative approach.
Background technology
Genetically modified organism technology be known as " use biotechnology " the most rapidly; Continuous development along with biotechnology; Increasing transgenic plant are applied to agriculture field; Promote agriculture prodn, help increasing peasant income, opened up new approach for solving the food problem that global population growth brings.So-called transgenic plant (Genetically modified organism; GMO) be meant and utilize biotechnology; To from different biologies, separate or the gene of synthetic is recombinated external, import the recipient plant cell then, and make new gene in recipient cell, integrate, express, its regeneration plant can be through asexual or sexual breeding; Foreign gene is entailed the offspring, and thus obtained genetically modified plant type is called transgenic plant.Directly be food or be that the food of raw material processing is called genetically modified foodGMF with transgenic plant.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; Satisfy human various demands, for example improve output, express the special nutrition composition even obtain the ability of the antiviral or pest-resistant evil of antiweed.For example genetically engineered soybean is the maximum genetically modified crops of whole world cultivated area; Genetically engineered soybean mainly is 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 the herbicide-tolerant Glyphosate 62 IPA Salt, make things convenient for soybean farming production and management greatly.
But along with the extensive plantation of genetically modified crops in worldwide, its edible safety and environmental safety problem have also caused consumers in general and national governments and associated mechanisms personnel's attention.Genetically modified crops produce in the laboratory, are the metastatic genes of large span, great-jump-forward, are different from the 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 secular 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 the 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 has become the main detection method of transgenic plant and products thereof owing to reasons such as detection sensitivity are high, applied widely, easy and simple to handle.Nucleic acid also is not easy to be destroyed in product processing owing to content in biomass cells is relatively stable relatively in addition, the detection target of doing transgenic plant and products thereof therefore commonly used.
Fluorescent quantitative poly Kettenreaktion (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 present detection of nucleic acids of generally acknowledging.The polymerase chain reaction is claimed the outer-gene amplification technique again, is the Protocols in Molecular Biology of the specific dna fragmentation of external at short notice a large amount of amplification.(real-time fluorescent quantitative PCR, FQ-PCR) technology has realized the leap of PCR by qualitative to quantitative for the nucleic acid quantification round pcr, the especially real-time fluorescence quantitative PCR that occur in recent years.The real-time fluorescence quantitative PCR technology adds the fluorophor of ability specific mark PCR product in the PCR reaction system, utilize the fluorescent signal accumulation whole PCR process of monitoring in real time, through typical curve unknown template is carried out quantitative analysis at last.The real-time fluorescence quantitative PCR technology is a kind of nucleic acid quantification and the analytical technology that on the qualitative technical foundation of PCR, grows up; This technology has been mixed together the high sensitivity of PCR, the high specific of DNA hybridization technique and the quantitative advantage of high precision of spectroscopic techniques; And easy and simple to handle, pollute fewly, become the important tool in many researchs such as molecular biology and the applied science.
In actual detected, reference material is very important.At present, domesticly be used for reference material that genetically modified organism detects also seldom, have only a kind of genetically engineered soybean powder reference material at present.Biometric unit leading in the world or laboratory have begun to carry out the development of transgenic quantitative PCR detection with reference material; 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 article that relate to 14 kinds of genetically modified crops kind more than 60; These standard substance are sold on market; But holding at high price of international certified reference material can not be satisfied the demand that increasing transgenic detects.And; Mostly existing these reference materials are the dried powder of histoorgans such as plant or the seed of original agricultural-food vegetable material; In use also need therefrom extract DNA, preparation, storage and the influence of mensuration process factors are numerous, are difficult to keep the constant amount; Be difficult to guarantee measuring stability, traceability is poor.
By contrast, the plasmid molecule reference material has clear superiority aspect a lot, and plasmid molecule is meant a kind of linearizing recombinant plasmid molecule that contains the specific fragment of transgenic testing goal foreign gene and internal standard gene.The empirical tests plasmid control molecule is good standard positive material surrogate in the GMO identification and detection.The advantage of plasmid molecule mainly is to cultivate in a large number through mikrobe, 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 use a lot of test experience chambers lacks strict traceability and investigates; Therefore detected result consistence, interoperability and safety can not guarantee; Quantitatively bring bigger uncertainty in give detecting, restricted the magnitude tracing development of China's transgenic product testing.
Summary of the invention
Technical problem to be solved by this invention is for the difficult problem that positive article lack and the positive criteria article dispose in the nucleic acid quantification PCR detection that overcomes existing genetically engineered soybean GTS40-3-2, and the plasmid control molecule of a kind of genetically engineered soybean GTS40-3-2 is provided.
The inventor inserts analysis of gene sequences through the external source to genetically engineered soybean strain GTS 40-3-2-3-2, and 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 through molecular cloning is building up to and forms artificial recombination plasmid molecule pXL02 in the plasmid molecule; 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 that makes up among the present invention can substitute genetically engineered soybean strain GTS 40-3-2-3-2 positive criteria article fully; This plasmid control molecule is applicable to genetically engineered soybean strain GTS 40-3-2-3-2 structures of samples specificity, strain specificity quantitative PCR analysis and detection fully; Thoroughly solve the difficult problem of standard material want in genetically engineered soybean strain GTS 40-3-2-3-2 detection, 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, contain the specific fragment of structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of genetically engineered soybean GTS40-3-2.
Among the present invention; The structure specific sequence of described genetically engineered soybean GTS40-3-2; Be meant that external source is inserted segmental one section sequence in the genetically engineered soybean GTS40-3-2 genome; This section sequence can be used as the structure specific sequence of genetically engineered soybean GTS40-3-2, the nucleotide fragments that the preferable sequence like CTP4 sequence in its genome and CTP4 sequence both sides is formed, and its preferred form can be part 35S promoter sequence, CTP4 sequence and portion C P4EPSPS sequence.Wherein, described " part " length that is 50-500bp.This " part " sequence has been arranged, can form the structure specific sequence of genetically engineered soybean GTS40-3-2.Its more preferred form is the nucleotide fragments that the CP4 EPSPS sequence of 109bp long 35S promoter sequence, CTP4 sequence (long altogether 216bp) and 365bp is formed.
The strain specificity sequence of described genetically engineered soybean GTS40-3-2; Be meant one section sequence in the genetically engineered soybean GTS40-3-2 genome; This section sequence can be used as the strain specificity sequence of genetically engineered soybean GTS40-3-2, and is preferable like exogenous insertion vector sequence that contains in its genome and the fragment of being formed with the adjacent soybean gene group sequence of this exogenous insertion vector.The form of the strain specificity sequence preference of said genetically engineered soybean GTS40-3-2 can be part exogenous insertion vector sequence and the nucleotide fragments formed with the adjacent part soybean gene group sequence of this exogenous insertion vector.Wherein, described " part " length that is 50-500bp.This " part " sequence has been arranged, can form the strain specificity sequence of genetically engineered soybean GTS40-3-2.The strain specificity sequence more preferred form of said genetically engineered soybean GTS40-3-2 is carrier sequence and 127bp and the adjacent soybean gene group sequence of this carrier sequence that comprises 192bp.
Among the present invention, described soybean internal standard gene Lectin refers to the soybean lectin plain gene, and it is conservative at soybean gene group camber, specificity between having kind, the characteristics of non-specific and single copy number in planting.The specific fragment of soybean internal standard gene Lectin described in the present invention is meant the fragment that has the soybean agglutinin gene specific in the soybean lectin plain gene.In the 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 preferably is exactly the nucleotide fragments that is selected from the 1029th to the 1579th successive 50-550bp of Lectin full-length gene, and optimum is exactly the 1029th to the 1579th fragment 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 may further comprise the 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 structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of the genetically engineered soybean GTS40-3-2 that successively pcr amplification is obtained respectively is cloned on the cloned plasmids carrier, obtains plasmid control molecule pXL02.
Among the present invention, the structure specific sequence of described genetically engineered soybean GTS40-3-2 is part 35S promoter sequence, CTP4 sequence and portion C P4EPSPS sequence; The strain specificity sequence of described genetically engineered soybean GTS40-3-2 is genetically engineered soybean GTS40-3-2 exogenous insertion vector and the adjacent sequence of soybean gene group; Described soybean internal standard gene Lectin is the soybean lectin plain gene.
Among the present invention, described PCR primer is that length is the oligonucleotide chain of 28 ± 10nt, and the specific fragment of structure specific sequence, strain specificity sequence or the soybean internal standard gene Lectin of itself and genetically engineered soybean GTS40-3-2 is identical or complementary.
Among 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 that designs.
Among 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 the plasmid vector according to the restriction enzyme digestion sites of design successively, obtains standard plasmid molecule pXL02.
Among the present invention, described plasmid vector can be any conventional carrier, preferably cloning vector, and better is the cloning vector that can in intestinal bacteria, breed; Like 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 may further comprise the 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 the plasmid control molecule pXL02 molecule;
3. prepare the phosphorus standardized solution of gradient concentration, and make the typical curve of inductively coupled plasma emission mass spectrum (ICP-MS) with this standardized solution.
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 phosphorus content and step of the plasmid control molecule pXL02 solution of gained of step, calculate the concentration of plasmid control molecule pXL02.
Among the present invention, the parametric optimization RF power of inductively coupled plasma emission mass spectrometric detection is 1100W, and the atomization gas flow is 1.08L/min, and the substreams amount is 1.2L/min, and the 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 make up 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 emission mass spectrum (ICP-MS) that it is carried out quantitatively having guaranteed the traceability that it is good.Plasmid control molecule of the present invention can substitute the positive criteria material of commentaries on classics because of soybean GTS40-3-2, is used for the quantitative PCR detection of genetically engineered soybean GTS40-3-2 structure specificity, strain specificity.
Description of drawings
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 usefulness.This plasmid standard molecule can substitute commentaries on classics because of soybean GTS40-3-2 positive criteria material, is used for the quantitative PCR detection of genetically engineered soybean GTS40-3-2 structure specificity, strain specificity.
The present invention inserts analysis of gene sequences through the external source to genetically engineered soybean GTS40-3-2, and design PCR primer is through the specific fragment of pcr amplification acquisition its structure specific sequence, strain specificity sequence and soybean internal standard gene Lectin; Method through molecular cloning is building up to it in 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 makes up can substitute genetically engineered soybean GTS40-3-2 positive criteria material fully; This plasmid standard molecule is applicable to genetically engineered soybean GTS40-3-2 structures of samples specificity, strain specificity quantitative PCR analysis and detection fully; Thoroughly solve the difficult problem that positive reference material lacks in the genetically engineered soybean GTS40-3-2 detection, and guaranteed the traceability of plasmid control molecule.
Nucleic acid quantification PCR detects the principle of soybean GTS-40-3-2
But adopt structure gene or the primer of strain specificity gene and soybean Lectin gene and the probe of two ends mark fluorescent among real-time fluorescence PCR technology and the specific amplification genetically engineered soybean GTS-40-3-2; Difference amplification assay sample DNA, and monitor the PCR product in real time.Meanwhile, with the positive criteria material (or positive criteria molecule) of identical primer, probe and condition amplification concentration known, to obtain stable typical curve.Can calculate the absolute content (copy number or concentration) of corresponding gene in the sample respectively according to the typical curve of foreign gene (structure specific gene or strain property specific gene) and native gene, and calculate the relative content of genetically engineered soybean GTS-40-3-2 in specimen by absolute content.As adopt positive criteria to divide the period of the day from 11 p.m. to 1 a.m to calculate relative content and should use gain factor.
Reference material
Reference material is to have one or more enough all even fine characteristic values of having confirmed, in order to correcting device, estimates measuring method or gives the material or the material of material assignment.
The transgenic plant detection reference material comprises standard positive material and plasmid control molecule.
The standard positive material is one type of reference material of processing with the primordial plant material, is the transgenic plant material that isozygotys, and in the PCR testing process, is used as the standard substance of positive control and configuration detection by quantitative, is used for the GMO mixed-level is quantized and analyzes.In transgenic quantitative PCR detection process, the positive criteria article are necessary, so the configuration requirement of positive criteria article is very strict, must can be applied to the transgenic quantitative PCR detection through a plurality of breadboard experimental verifications.This type derives from the reference material of agricultural-food, and its preparation, storage and mensuration process are influenced by a lot of factors, are difficult to keep the constant amount, and is not that all crop transgenic strains all exist the standard positive material.
Plasmid control molecule
Plasmid control molecule is meant a kind of linearizing recombinant plasmid molecule that contains the specific fragment of transgenic testing goal foreign gene and internal standard gene.The empirical tests plasmid control molecule is good standard positive material surrogate in the GMO identification and detection.The advantage of plasmid molecule mainly is to cultivate in a large number through mikrobe, 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 the scholar plasmid control molecule to be called " gold standard material ".In transgenic quantitative PCR detection process, standard molecule converts according to molecular weight size and plant genome DNA molecule, thereby accomplishes the quantitative analysis to the transgenic sample.Standard molecule can be applied to transgenic PCR and detect, especially in the quantitative PCR detection.
Plasmid control molecule of the present invention contains the specific fragment of structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of genetically engineered soybean GTS40-3-2.
Among the present invention; The structure specific sequence of described genetically engineered soybean GTS40-3-2; Be meant that external source is inserted segmental one section sequence in the genetically engineered soybean GTS40-3-2 genome; This section sequence can be used as the structure specific sequence of genetically engineered soybean GTS40-3-2, the nucleotide fragments that the preferable sequence like CTP4 sequence in its genome and CTP4 sequence both sides is formed, and its preferred form can be part 35S promoter sequence, CTP4 sequence and portion C P4 EPSPS sequence.Wherein, described " part " length that is 50-500bp.This " part " sequence has been arranged, can form the structure specific sequence of genetically engineered soybean GTS40-3-2.Its more preferred form is the nucleotide fragments that the CP4 EPSPS sequence of 109bp long 35S promoter sequence, CTP4 sequence (long altogether 216bp) and 365bp is formed.The structure specific sequence of described genetically engineered soybean GTS40-3-2 preferable shown in SEQ ID NO.1, wherein the 1st to 109 is part 35S promoter sequence, the 110th to 326 is the CTP4 sequence, the 327th to 691 is portion C P4 EPSPS sequence.
The strain specificity sequence of genetically engineered soybean GTS40-3-2 of the present invention; Be meant one section sequence in the genetically engineered soybean GTS40-3-2 genome; This section sequence can be used as the strain specificity sequence of genetically engineered soybean GTS40-3-2, and is preferable like exogenous insertion vector sequence that contains in its genome and the fragment of being formed with the adjacent soybean gene group sequence of this exogenous insertion vector.The form of the strain specificity sequence preference of said genetically engineered soybean GTS40-3-2 can be part exogenous insertion vector sequence and the nucleotide fragments formed with the adjacent part soybean gene group sequence of this exogenous insertion vector.Wherein, described " part " length that can be 20-500bp.This " part " sequence has been arranged, can form the strain specificity sequence of genetically engineered soybean GTS40-3-2.The strain specificity sequence more preferred form of said genetically engineered soybean GTS40-3-2 is carrier sequence and 127bp and the adjacent soybean gene group sequence of this carrier sequence that comprises 192bp.Preferably, the strain specificity sequence of described genetically engineered soybean GTS40-3-2 is shown in SEQ ID NO.2, and wherein the 1st to the 192nd is the exogenous insertion vector sequence, and wherein the 193rd to the 319th is adjacent soybean gene group sequence.
Among the present invention, described soybean internal standard gene Lectin refers to the soybean lectin plain gene, and it is conservative at soybean gene group camber, specificity between having kind, the characteristics of non-specific and single copy number in planting.The specific fragment of soybean internal standard gene Lectin described in the present invention is meant the fragment that has the soybean agglutinin gene specific in the soybean lectin plain gene.In the soybean lectin plain gene, the 1029th～the 1579th fragment of full-length gene be have specific.The specific fragment of soybean internal standard gene Lectin of the present invention preferably is exactly the nucleotide fragments that is selected from the 1029th to the 1579th successive 50-550bp of Lectin full-length gene, and optimum is exactly the 1029th to the 1579th fragment of Lectin full-length gene.Preferably, the sequence of the specific fragment of described soybean internal standard gene Lectin is shown in SEQ ID NO.3.
The sequence of plasmid control molecule pXL02 of the present invention preferable shown in SEQ ID NO.10; Wherein the 1336th is the structure specific sequence to the 2026th; The 986th to the 1305th is the strain specificity sequence, and the 413rd is the specific fragment of internal standard gene Lectin to the 963rd.
The plasmid control molecule construction process
Plasmid control molecule pXL02 construction process according to the invention may further comprise the steps:
1. utilize DB (detecting assessment share 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 such as Genbank, Shanghai City genetically modified organism security.
2. design the PCR Auele Specific Primer;
Described PCR primer refers to the oligonucleotide chain that length is 28 ± 10nt, and the specific fragment of structure specific sequence, strain specificity sequence or the soybean internal standard gene Lectin of itself and genetically engineered soybean GTS40-3-2 is identical or complementary.
Preferable, the primer centering of the structure specific sequence of amplification genetically engineered soybean GTS40-3-2, the sequence of a primer is SEQ ID NO.4, another is SEQ ID NO.5.
The primer centering of the strain specificity sequence of amplification genetically engineered soybean GTS40-3-2, the sequence of a primer is SEQ ID NO.6, another is SEQ ID NO.7.
The primer centering 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, 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 structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of the genetically engineered soybean GTS40-3-2 that successively pcr amplification is obtained respectively is cloned on the 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 the plasmid vector according to the restriction enzyme digestion sites that designs successively, obtains standard plasmid molecule pXL02.
Described plasmid vector can be any conventional carrier, preferably cloning vector, and better is the cloning vector that can in intestinal bacteria, breed, like 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; Be meant and detect the characteristics such as specificity, sensitivity, repeatability and repeatability of plasmid control molecule pXL02 when carrying out quantitative PCR analysis; Identifying 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 according to the invention may further comprise the steps:
1. extract plasmid control molecule pXL02;
2. according to based composition and the sequence length of plasmid control molecule pXL02, calculate the content of the phosphoric of plasmid control molecule pXL02;
3. prepare the phosphorus standardized solution of gradient concentration, and make the typical curve of ICP-MS with this standardized solution.
4. ICP-MS detects plasmid control molecule pXL02, and draws the phosphorus content of plasmid control molecule pXL02 according to typical curve.
5. calculate the concentration of plasmid control molecule pXL02 according to the phosphorus content of plasmid control molecule pXL02.
Elaborate in the face of embodiment of the present invention down.Present embodiment is implemented under the prerequisite of technical scheme of the present invention, has provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.The experiment side of unreceipted actual conditions fails in the following example; Usually operate according to normal condition; As can write molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press with reference to Sambrook etc.; 1989) described condition, or the condition of advising according to manufacturer." room temperature " described in the present invention is meant the temperature of the operation room that makes an experiment, 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 available from the white good development in science and technology in Shanghai ltd.
PUC19 carrier, Taq archaeal dna polymerase, T4DNA ligase enzyme, dNTP, DL2000 Marker
Available from the white good development in science and technology in Shanghai ltd;
Dna primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
DY-501 type nucleic acid electrophoresis apparatus (Shanghai Precision Scientific Apparatus Co., Ltd)
PTC-100 type pcr amplification appearance (MJ Research Inc.)
DNA electrophoretic analysis system comprises camera bellows, digital camera, computingmachine, scanner, ink-jet printer, photosensitive stamping machine, Tanon UV-2000 uv analyzer, Gis gel images analysis software (sky, Shanghai ability company)
Other Instruments comprises: whizzer, thermostat water bath, incubator, day equality.
Three, experimental technique and process
1, search soybean internal standard gene Lectin in GenBank; The genetically modified organism security detects assessment share service platform (http://www.shgmo.org/welcome.htm) and goes up concrete inserted mode and the copy number of exogenous insertion vector in genetically modified crops that inquiry obtains genetically engineered soybean GTS40-3-2 in Shanghai City, and consults the sequence information of main element in the exogenous insertion vector.
2, make up the PCR primer sequence design of plasmid control molecule pXL02
The synoptic diagram that makes up plasmid control molecule pXL02 is seen Fig. 1.According to the genetically engineered soybean GTS40-3-2 external source insertion sequence information that obtains, utilize two pairs of PCR primers of software Primer 5.0 designs.A pair of primer be used to the to increase structure specific gene of genetically engineered soybean GTS40-3-2, a primer is positioned on the 35S promoter sequence, and one is positioned on the CP4 EPSPS gene order; Another is to primer be used to the to increase strain specificity gene of genetically engineered soybean GTS40-3-2, and a primer is positioned on the soybean gene group, and another is positioned on the exogenous insertion vector of genetically engineered soybean GTS40-3-2.Design a pair of primer soybean internal standard gene Lectin specific sequence that is used to increase in addition.The two ends of each primer add restriction enzyme site and the protection base that molecular cloning is required.
In the design of primers process; Make every effort to make the plasmid of structure to be applicable to the detection of more genetically engineered soybean GTS40-3-2; The sequence that comprises in the plasmid both applicable to the amplification of structure specific sequence, also applicable to the strain specificity sequence amplification, will be taken into account the specificity of detected result simultaneously.
Concrete primer sequence is seen table 1.
Table 1. makes up 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
Primer according to table 1; With genetically engineered soybean GTS40-3-2 genomic dna is template; Its structure specific sequence, strain specificity sequence and soybean internal standard gene order are carried out pcr amplification (reaction system and condition are seen table 2,3), obtain the structure specific sequence of 709bp, the strain specificity sequence of 337bp and the soybean internal standard Lectin gene order of 570bp.Amplified production is reclaimed purifying.
Table 2. makes up 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 unit/reaction) | 1 |
| Dna profiling | 1 |
| ddH 2O | Complement to 20 μ L |
Table 3. makes up the pcr amplification condition of plasmid control molecule pXL02
4, with the PCR product cloning to plasmid vector pUC19
The structure specific sequence and the carrier pUC19 that obtain with restriction enzyme HindIII digest amplification respectively; Reclaim structure specific sequence and linear plasmid pUC19 after enzyme is cut; The T4 ligase enzyme connects, and connects product transformed into escherichia coli DH5 α, plasmid molecule pUC19_1 in the middle of obtaining.The strain specificity sequence and the middle plasmid molecule pUC19_1 that obtain with restriction enzyme XbaI digest amplification respectively; Interstitial granules pUC19_1 in strain specificity sequence after the recovery enzyme is cut and the linearity; The T4 ligase enzyme connects; Connect product transformed into escherichia coli DH5 α, plasmid molecule pUC19_2 in the middle of obtaining.The Lectin gene specific sequence and the middle plasmid molecule pUC19_2 that obtain with restriction enzyme KpnI digest amplification; Interstitial granules pUC19_2 in Lectin sequence after the recovery enzyme is cut and the linearity; The T4 ligase enzyme connects; Connect product transformed into escherichia coli DH5 α, obtain plasmid control molecule pXL02, see Fig. 2.Endonuclease reaction system and linked system are seen table 4,5.
Table 4. endonuclease reaction system
| Reaction reagent | Consumption (μ L) |
| 10×buffer | 2 |
| Restriction enzyme | 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 |
| The T4 ligase enzyme | 2 |
| Dna fragmentation | 6 |
| Plasmid vector | 7 |
| ddH 2O | Complement to 20 μ L |
Experimental example 2 usefulness ICP-MS carry out quantitatively the plasmid control molecule pXL02 that makes up
One, experiment reagent
Phosphate radical in GBW (E) 081216 water-phosphorus composition analytical standard material 0.100g/L.
Two, laboratory apparatus
Inductively coupled plasma emission mass spectrograph (Perkin elmer ELAN DRC-e).
Three, experimental technique and process
1,, calculates the content of the phosphoric of plasmid control molecule pXL02 according to based composition and the sequence length of plasmid control molecule pXL02;
2, the ICP-MS experiment parameter is: RF power is 1100W, and the atomization gas flow is 1.08L/min, and the substreams amount is 1.2L/min, and the isoelectronic species airshed is 16L/min.
3, the phosphorus standardized solution (0,10,50,100,200ppb) of preparation gradient concentration, and make the typical curve of ICP-MS with this standardized solution;
4, ICP-MS detects plasmid control molecule pXL02, and draws the phosphorus content of plasmid control molecule pXL02 according to typical curve;
5, calculate the concentration of plasmid control molecule pXL02 according to the phosphorus content of plasmid control molecule pXL02;
Four, experimental result
According to the based composition of pXL02, calculate and learn that phosphorus content wherein is about 10.197%; According to the result of ICP-MS, the phosphorus content that records among the plasmid control molecule pXL02 of this batch extraction is 15.957ppm, learns after the conversion that the concentration of this batch plasmid control molecule pXL02 is about 3.63 * 10
10Copy/μ L.
The application of plasmid control molecule pXL02 in actual detected that embodiment 3 makes up
One, experiment reagent
Plant genome DNA extracts and purifying adopts the plant genome DNA of OMEGA company to extract test kit.
DNA extracts and purifying adopts the DNA of OMEGA company to extract test kit.
PUC19 carrier, T4DNA ligase enzyme and damping fluid thereof, dNTPs, Taq archaeal dna polymerase and damping fluid thereof, DNA Marker are available from the white good development in science and technology in Shanghai ltd.
Primer and probe are given birth to worker's biotechnology ltd by Shanghai and are synthesized.
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
DY-501 type nucleic acid electrophoresis apparatus (Shanghai Precision Scientific Apparatus Co., Ltd).
Opticon-2 quantitative pcr amplification appearance (MJ Research Inc.).
DNA electrophoretic analysis system comprises camera bellows, digital camera, computingmachine, scanner, ink-jet printer, photosensitive stamping machine, Tanon UV-2000 uv analyzer, Gis gel images analysis software (sky, Shanghai ability 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 an amount of soybean sample, add liquid nitrogen grind into powder in mortar, take by weighing about 10-50mg powder to the 1.5mL centrifuge tube, add 800 μ L Buffer P1;
B, 65 ℃ of incubations 10 minutes, mixing is twice therebetween;
C, adding 140 μ L Buffer P2, the vibration mixing.Centrifugal 10 minutes of >=10000 * g carefully moves to supernatant in the one new pipe;
The Virahol of d, 0.7 times of volume of adding, vibration is with deposit D NA, and 10000 * g is centrifugal two minutes immediately;
E, supernatant discarded add 300 μ L and are preheated to 65 ℃ sterilized water, and vibration is with resuspended DNA.Add 4 μ L RNase A mixings;
F, adding 150 μ L Buffer P3 then add 300 μ L absolute ethyl alcohols, the vibration mixing;
G, all samples joined in the HiBind DNA adsorption column (place the 2mL collection tube), centrifugal one minute of 10000 * g is to combine DNA.Discard collection tube and liquid wherein;
H, adsorption column is put into new collection tube, wash with 650 μ L DNA Wash Buffer (absolute ethyl alcohol dilution);
I, repeated washing step are washed with 650 μ L DNA Wash Buffer (absolute ethyl alcohol dilution);
J, maximum speed (be no more than 20000 * g) centrifugal 2 minutes with dry adsorption column;
K, adsorption column is put into a clean 1.5mL pipe, add 100 μ L and be preheated to 65 ℃ Elution Buffer (or 10mM Tris buffer pH8.5/9.0 or aseptic deionized water), room temperature was placed 3-5 minute.Centrifugal 1 minute eluted dna of 10000 * g.
2) extraction of DNA
A, with the bacterium of 1.5-5.0mL incubated overnight centrifugal 1 minute, supernatant discarded in room temperature 10000 * g;
B, adding 250 μ L Solution I (containing RNase A), abundant mixing vibrates;
C, adding 250 μ L Solution II, the gentle mixing that turns upside down, room temperature is placed 2 minutes so that the abundant cracking of thalline;
D, adding 350 μ L Solution III fully put upside down mixing up and down for several times immediately, until forming uniform white precipitate;
E, >=centrifugal 10 minutes of 13000 * g room temperature;
F, supernatant is moved in the clean HiBind adsorption column (placing the 2mL collection tube) carefully centrifugal 1 minute of 10000 * g room temperature;
G, discard the liquid in the collection tube, add 500 μ L Buffer HB and clean adsorption column, centrifugal 1 minute of 10000 * g room temperature;
H, discard the liquid in the collection tube, add 700 μ L DNA Wash Buffer (absolute ethyl alcohol dilution) and clean adsorption column;
I, repeat above-mentioned cleaning step;
Centrifugal 2 minutes of j, 13000 * g are with dry adsorption column;
K, adsorption column is placed a clean 1.5mL pipe, add 30 μ L-50 μ L Elution Buffer (or 10mM Tris buffer pH8.5) or sterilization deionized water, room temperature was placed 1-2 minute, and centrifugal 1 minute of 13000 * g is with eluted dna.
2, plasmid control molecule pXL02 is used for the limit of detection (LOD) and the quantitation limit (LOQ) of quantitative PCR detection
PCR detection architecture and reaction conditions among the employing standard GB/T 19495.5-2004 " transgenic product detect 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 the LOD and the LOQ of standard substance tests quantitative PCR detecting method.Each reacts triplicate, according to the typical curve of quantitative pcr amplification and the linear relationship between the amplification fluorescent signal, and when confirming to use the plasmid control molecule pXL02 replacing positive reference material that makes up, the LOD of this quantitative PCR detecting method and LOQ.
3, the repeatability and the repeatability of plasmid control molecule pXL02 detection by quantitative system
With PCR detection architecture and the reaction conditions among the standard GB/T 19495.5-2004 " transgenic product detect 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 the test of repeatability and repdocutbility, each reacts triplicate.According to the typical curve of quantitative pcr amplification, confirm repeatability and repeatability according to the genetically engineered soybean GTS40-3-2 quantitative PCR reaction of plasmid control molecule pXL02 foundation.
4, use the quantitative analysis of plasmid control molecule pXL02 to actual genetically engineered soybean GTS40-3-2 sample
With PCR detection architecture and the reaction conditions among the standard GB/T 19495.5-2004 " transgenic product detects nucleic acid quantification PCR detection method "; According to the quantitative PCR typical curve of drawing; To transgenic content is that 10% genetically engineered soybean GTS40-3-2 biased sample is analyzed, and confirms whether the genetically engineered soybean GTS40-3-2 detection by quantitative plasmid control molecule that makes up 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 the limit of detection (LOD) and the quantitation limit (LOQ) of quantitative PCR detection
PCR detection architecture and reaction conditions among the employing standard GB/T 19495.5-2004 " transgenic product detect 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 the LOD and the LOQ of standard substance tests quantitative PCR detecting method.Confirm that through 3 repeated experiments the LOD of this system is 3 copies, LOQ is 30 copies.Explain that the pXL02 plasmid control molecule that makes up can replace genetically engineered soybean GTS40-3-2 positive criteria article 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
PCR detection architecture and reaction conditions among the employing standard GB/T 19495.5-2004 " transgenic product detect 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) carries out the test of repeatability and repdocutbility; Between 3 parallel reactors with 3 different repeated experiments between the Ct value standard deviation that obtains basically all less than 0.2; Explanation is good according to the repeatability and the repeatability of the genetically engineered soybean GTS40-3-2 quantitative PCR reaction that the pXL02 plasmid control molecule is set up, and can be used for the further quantitative analysis of actual genetically engineered soybean GTS40-3-2 sample.
3, use the quantitative analysis of plasmid control molecule pXL02 to actual genetically engineered soybean GTS40-3-2 sample
With PCR detection architecture and the reaction conditions among the standard GB/T 19495.5-2004 " transgenic product detects nucleic acid quantification PCR detection method "; According to the quantitative PCR typical curve of drawing; To transgenic content is that 10% genetically engineered soybean GTS40-3-2 biased sample is analyzed; Quantitative analysis results shows that the transgenic content of this sample is 11.54%; With the deviation of actual value be 15.4%, the deviation of detected result shows that plasmid control molecule pXL02 that the present invention makes up is applicable to quantitative PCR analysis and the detection of genetically engineered soybean GTS40-3-2 in the 0-0.25 scope that ISO genetically modified foodGMF examination criteria allows.
Should be understood that after having read foregoing of the present invention those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the plasmid control molecule of a genetically engineered soybean GTS40-3-2 is characterized in that, contains the specific fragment of structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of genetically engineered soybean GTS40-3-2.
2. the plasmid control molecule of genetically engineered soybean GTS40-3-2 as claimed in claim 1; It is characterized in that the structure specific sequence of described genetically engineered soybean GTS40-3-2 is the nucleotide fragments that part 35S promoter sequence, CTP4 sequence and portion C P4 EPSPS sequence are formed; The strain specificity sequence of described genetically engineered soybean GTS40-3-2 is genetically engineered soybean GTS40-3-2 exogenous insertion vector and the nucleotide fragments formed with the adjacent soybean gene group sequence of this exogenous insertion vector, and described " part " is the length of 50-500bp; It is characterized in that described soybean internal standard gene Lectin is the soybean lectin plain gene.
3. the preparation method of the plasmid control molecule of a genetically engineered soybean GTS40-3-2 according to claim 1 or claim 2 is characterized in that, may further comprise the 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 structure specific sequence, strain specificity sequence and the soybean internal standard gene Lectin of the genetically engineered soybean GTS40-3-2 that successively pcr amplification is obtained respectively is cloned on the 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 the structure specific sequence of described genetically engineered soybean GTS40-3-2 is the nucleotide fragments that part 35S promoter sequence, CTP4 sequence and portion C P4 EPSPS sequence are formed; The strain specificity sequence of described genetically engineered soybean GTS40-3-2 is genetically engineered soybean GTS40-3-2 exogenous insertion vector and the nucleotide fragments formed with the adjacent soybean gene group sequence of this exogenous insertion vector; Described soybean internal standard gene Lectin is the soybean lectin plain gene.
5. the preparation method of the plasmid control molecule of genetically engineered soybean GTS40-3-2 detection by quantitative 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 that designs.
6. the preparation method of the plasmid control molecule of genetically engineered soybean GTS40-3-2 detection by quantitative 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 the plasmid vector successively, obtains standard plasmid molecule pXL02.
7. the quantivative approach of the plasmid control molecule of a genetically engineered soybean GTS40-3-2 according to claim 1 or claim 2 is characterized in that, may further comprise the 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 the plasmid control molecule pXL02 molecule;
3. prepare the phosphorus standardized solution of gradient concentration, and make the typical curve of inductively coupled plasma emission mass spectrum (ICP-MS) with this standardized solution.
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 phosphorus content and step of the plasmid control molecule pXL02 solution of gained of step, calculate the concentration of plasmid control molecule pXL02.
8. the quantivative approach of the plasmid control molecule of described genetically engineered soybean GTS40-3-2 as claimed in claim 7; It is characterized in that the ICP-MS parameter is following: RF power is 1100W, and the atomization gas flow is 1.08L/min; The substreams amount is 1.2L/min, and the isoelectronic species airshed is 16L/min.
9. the nucleic acid quantification PCR detection method of a genetically engineered soybean GTS40-3-2 is characterized in that, institute's accepted standard material is a plasmid control molecule according to claim 1 or claim 2.
10. according to claim 1 or claim 2 the application of plasmid control molecule in the nucleic acid quantification PCR of genetically engineered soybean GTS40-3-2 detects.
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