CN103146824A - Recombinant standard plasmid and kit for PCR (Polymerase Chain Reaction) detection of transgenic rice - Google Patents
Recombinant standard plasmid and kit for PCR (Polymerase Chain Reaction) detection of transgenic rice Download PDFInfo
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Abstract
The invention provides a recombinant standard plasmid for qualitative and quantitative PCR (Polymerase Chain Reaction) detection of transgenic rice KF6, transgenic rice TT51, and transgenic rice KMD1. The recombinant standard plasmid is obtained by the following method: orderly joining KF6 flanking sequence shown in SEQ ID NO.1, TT51 flanking sequence shown in SEQ ID NO.2, KMD1 flanking sequence shown in SEQ ID NO.3, and PLD gene shown in SEQ ID NO.4 to obtain a fusion segment, and cloning the obtained fusion segment into a carrier pMD-18T to obtain the recombinant standard plasmid, which is labeled as pMD-KTK. The recombinant standard plasmid can replace positive standard substances of the transgenic rice KF6, the transgenic rice TT51, and the transgenic rice KMD1 to be used for specific qualitative and quantitative PCR detection of the transformant of the transgenic rice KF6, the transformant of the transgenic rice TT51, and the transformant of the transgenic rice KMD1; the plasmid has the advantages of good specificity, high sensitivity and the like; when the recombinant standard plasmid is used for quantitative analysis of an actual sample, the deviation of the sample detection result is within an allowable range widely accepted in the world; and the problem that the positive standard substances of the transgenic rice KF6, the transgenic rice TT51, and the transgenic rice KMD1 are in shortage in a detection process is well solved.
Description
(1) technical field
The present invention relates to the restructuring standard plasmid that a kind of quantitative and qualitative analysis for three kinds of transgenic paddy rice KF6, TT51, KMD1 detects, and the test kit that contains this restructuring standard plasmid.
(2) background technology
From the first transgenic Fructus Lycopersici esculenti " FLAVR SAVR " in 1994 since U.S. approval is commercially produced, along with transgenic technology develops rapidly, many valuable foreign genes have imported in plant, and increasing genetically modified crops go through in country variant and regional commercialization plantation.For paddy rice, there is at present the transgenic paddy rice of many pest-resistant, disease-resistant, herbicide-resistants and nutritive ingredient improvement to research and develop successfully, transgenic paddy rice strain: KF6, TT51 and KMD1 are the pest-resistant Bt paddy rice that China has independent intellectual property right, wherein transgenic pest-resistant rice China extensive No. 1 (TT51) was in 2009, and China Ministry of Agriculture has issued its safety certificate in Hubei Province's production application.
for ensureing the interests in the agricultural-food foreign trade, various countries set up the transgenic labeling system in succession, accuracy and sensitivity to the transgenosis detection technique have proposed strict requirement, at present the detection of genetically modified crops and products thereof mainly contained protein level detection and nucleic acid level detection, and due to the extraction of nucleic acid, the advantage of the aspects such as preservation and stability, make the transgenosis detection technique based on detection of nucleic acids be rapidly developed, wherein get the fastest with polymerase chain reaction (PCR) technical development, genetically modified crops PCR detection method is divided into again 4 levels, being respectively examination detects, gene test, building specific detection and specificity of transformant detects, in daily testing, because the transgenosis sample size is larger, it is particularly important that genetically modified screening seems, and the shortage of positive criteria product can't satisfy the demand of screening in the Transgene-safty supervision, therefore developing a kind of the detection for the transgenic paddy rice examination has great importance with plasmid control molecule.
The Objective Concept of positive plasmid molecule Japan scientist Kuribara H equals to propose in 2002, and it refers to a kind of linearizing recombinant plasmid molecule that contains the specific amplification fragment of purpose foreign gene that genetically modified crops detect and internal standard gene.The advantage of positive plasmid molecule is mainly to cultivate in a large number by microorganism, and processing ease, and same positive plasmid molecule can comprise a plurality of external source goal gene simultaneously, and extraction of plasmid DNA is easy and purity is higher.Genetically modified crops positive plasmid molecule is a kind of of transgenosis reference material.Exploitation positive plasmid molecule can effectively solve a difficult problem that lacks certified reference material and positive material in testing.
(3) summary of the invention
The purpose of this invention is to provide a kind of is rich No. 6 of three kinds of transgenic paddy rice KF6(sections), extensive No. 1 of TT51(China), the KMD1(Kemingdao) specificity of transformant detect with restructuring standard plasmid and application thereof, satisfy the demand in transgenic paddy rice security control examination testing.
The technical solution used in the present invention is:
The restructuring standard plasmid that a kind of quantitative and qualitative analysis PCR for transgenic paddy rice KF6, TT51, KMD1 detects, obtain by the following method: the PLD gene that the TT51 flanking sequence shown in the KF6 flanking sequence shown in SEQ ID No.1, SEQ ID No.2, the KMD1 flanking sequence shown in SEQ ID No.3 are connected with SEQ ID No.4 connects acquisition fusion fragment successively, gained being merged fragment is cloned in carrier pMD18-T again, obtain described restructuring standard plasmid, be designated as pMD-KTK.
KF6 flanking sequence following (length 454bp):
Gatcccgaaggccaacacaataggaccggaatcctatgatgttatcccatgctaatgtatccagagcgatggcttgctttgagcactctaatttcttcaaagtaacggcgccggaggcacgacccggccagttaaggccaggagcgcatctgatccaacttaataacacattgcggacgtttttaatgtactgaattaacgccgaattaattcgggggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatactaagggtttcttataggctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacattattatggagaaactcgagcttgtcgatcgacagatcccggtcggcatctactctatttctttgccctcggacga
TT51 flanking sequence following (length 305bp):
Ccggcgtcaatacgggataataccgcgccacatagcagaactttaacccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgatttgtagagagagactggtgatttcagcgggcatgcctgcaggtcgactctagaggatcccggacgagtgctggggcagataagcagtagtggtggggctacgaacatattccttttccttctggacgctaccactcatatgttccaaaattacaaatttgtcctttgtatttgttgcaattttcatgtaagaaatccaacgaggct
KMD1 flanking sequence following (length 398bp):
Tgtggagcgtcaactgggagcttatcttattactaattagtattaccattgccattacgtctgaaacagacggggctcccatttctttttgatgtcgttagtacagccggcttcgtttttcttttctggctgtctcggctgtcacctgtcagtctcgtcagcaactggggtgttccgccgacgagggcgccgtggacttcgcgggctcgcggcgatggcgatgcgcgctccaactgcggcgggtctcaccgggctgcgtgcgtacgccgatatgcctgcccatctcgggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactggggtggtttttcttttcaccagtgagacgggcaacagc
PLD gene order following (length 250bp):
Agaccagaagacattgagccgttgcatctgattcccagagagatttctctgaagattgtgaacaagattgaagctggtgagcgttttgcagtctatgttgtgctgccaatgtggcctgaaggacctcctgctagtggatcagtgcaggcaatactggattggcagaggaggacaatggagatgatgtactatgatattgccgttgcacttgaggcgaagaggatcaatgctgacccgagggattacctta。
The present invention is according to the specificity of transformant sequence of three transgenic paddy rice KF6, TT51, KMD1 and the sequence of paddy rice internal standard gene PLD, design corresponding primer, utilize overlapping pcr that four sequence fragments are merged, the recycling molecule clone technology will merge fragment and be cloned in the pMD18-T carrier, obtain plasmid molecule restructuring standard plasmid pMD-KTK.
Concrete, described fusion fragment nucleotide sequence is as shown in SEQ ID No.5 (length 1407bp):
gatcccgaaggccaacacaataggaccggaatcctatgatgttatcccatgctaatgtatccagagcgatggcttgctttgagcactctaatttcttcaaagtaacggcgccggaggcacgacccggccagttaaggccaggagcgcatctgatccaacttaataacacattgcggacgtttttaatgtactgaattaacgccgaattaattcgggggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatactaagggtttcttataggctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacattattatggagaaactcgagcttgtcgatcgacagatcccggtcggcatctactctatttctttgccctcggacgaccggcgtcaatacgggataataccgcgccacatagcagaactttaacccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgatttgtagagagagactggtgatttcagcgggcatgcctgcaggtcgactctagaggatcccggacgagtgctggggcagataagcagtagtggtggggctacgaacatattccttttccttctggacgctaccactcatatgttccaaaattacaaatttgtcctttgtatttgttgcaattttcatgtaagaaatccaacgaggcttgtggagcgtcaactgggagcttatcttattactaattagtattaccattgccattacgtctgaaacagacggggctcccatttctttttgatgtcgttagtacagccggcttcgtttttcttttctggctgtctcggctgtcacctgtcagtctcgtcagcaactggggtgttccgccgacgagggcgccgtggacttcgcgggctcgcggcgatggcgatgcgcgctccaactgcggcgggtctcaccgggctgcgtgcgtacgccgatatgcctgcccatctcgggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactggggtggtttttcttttcaccagtgagacgggcaacagcagaccagaagacattgagccgttgcatctgattcccagagagatttctctgaagattgtgaacaagattgaagctggtgagcgttttgcagtctatgttgtgctgccaatgtggcctgaaggacctcctgctagtggatcagtgcaggcaatactggattggcagaggaggacaatggagatgatgtactatgatattgccgttgcacttgaggcgaagaggatcaatgctgacccgagggattacctta。
Restructuring standard plasmid pMD-KTK of the present invention specifically can build as follows:
(1) utilize the retrieval of GenBank database and pertinent literature, obtain paddy rice internal standard gene PLD and transgenic paddy rice KF6, TT51, KMD1 specificity of transformant sequence separately.
Described paddy rice internal standard gene PLD refers to the paddy rice phospholipase D, and is conservative at the rice genome camber, has the characteristics such as non-specific in specificity between kind, kind and single copy number.
The specific sequence of conversion separately of described transgenic paddy rice KF6, TT51, KMD1 refers to the DNA sequence dna of corresponding transgenic paddy rice external source insertion sequence and rice genome joining region.
(2) according to the specific sequence of conversion separately of PLD gene order, transgenic paddy rice KF6, TT51, KMD1 (corresponding sequence as shown in Figure 2), design overlapping PCR primer, the genomic dna of transgenic paddy rice KF6, TT51, KMD1 positive criteria product is carried out pcr amplification.
Described overlapping PCR primer refers to the primer identical or complementary with the sequence that transforms separately specific sequence and paddy rice internal standard gene PLD of transgenic paddy rice KF6, TT51, KMD1, makes each purpose fragment reach seamless link.
Described transgenic paddy rice KF6, TT51 and KMD1 positive criteria product refer to that transgenosis content is 100% transgenic paddy rice KF6, TT51 and the positive criteria product powder of KMD1.
(3) specific amplification transgenic paddy rice KF6 transformant sequence.
Described specific amplification refers to utilize the specificity of transformant sequence of the primer amplified transgenic paddy rice KF6 of design, and primer is respectively:
KF6-F：GATCCCGAAGGCCAACACAATAGG；
KF6-R-add：tatcccgtattgacgccggTCGTCCGAGGGCAAAGAAATAGAG；
Specific amplification transgenic paddy rice TT51 transformant sequence.
Described specific amplification refers to utilize the specificity of transformant sequence of the primer amplified transgenic paddy rice TT51 of design, and primer is respectively:
TT51-F-add：ttctttgccctcggacgaCCGGCGTCAATACGGGATAAT；
TT51-R-add：ccagttgacgctccacaAGCCTCGTTGGATTTCTTACA；
Specific amplification transgenic paddy rice KMD1 transformant sequence.
Described specific amplification refers to utilize the specificity of transformant sequence of the primer amplified transgenic paddy rice KMD1 of design, and primer is respectively:
KM-F-add：aagaaatccaacgaggctTGTGGAGCGTCAACTGGGAGC；
KM-R-add：ctcaatgtcttctggtctGCTGTTGCCCGTCTCACTGGT；
Specific amplification paddy rice internal standard gene PLD sequence.
Described specific amplification refers to utilize the primer amplified paddy rice internal standard gene of design
The sequence of PLD, primer is respectively:
PLD-F-add：gtgagacgggcaacagcAGACCAGAAGACATTGAGCCG；
PLD-R：TAAGGTAATCCCTCGGGTCAG；
(4) splicing of the specificity of transformant sequence of the specificity of transformant sequence of transgenic paddy rice KF6 and transgenic paddy rice TT51.
Described splicing, refer to the PCR product of specificity of transformant sequence of the PCR product of transformant distinguished sequence of the transgenic paddy rice KF6 that will obtain and transgenic paddy rice TT51 jointly as template, utilize overlapping PCR that the distinguished sequence of KF6 and TT51 is linked together (KF6+TT51), the primer is:
KF6-F：GATCCCGAAGGCCAACACAATAGG；
TT51-R-add：ccagttgacgctccacaAGCCTCGTTGGATTTCTTACA；
The splicing of the specific sequence of the specificity of transformant sequence of transgenic paddy rice KMD1 and paddy rice internal standard gene PLD.
Described splicing refers to the PCR product of PCR product and PLD of specificity of transformant sequence of the transgenic paddy rice KMD1 that will obtain jointly as template, utilize overlapping PCR with KMD1 and
The distinguished sequence of PLD links together (KMD1+PLD), and the primer is:
KM-F-add：aagaaatccaacgaggctTGTGGAGCGTCAACTGGGAGC；
PLD-R：TAAGGTAATCCCTCGGGTCAG；
The splicing of complete recombinant fragment.
Described splicing, refer to (KF6+TT51) that will obtain again with (KMD1+PLD) product jointly as template, utilize overlapping PCR that they are linked together, complete
The fusion of KF6+TT51+KMD1+PLD connects, and the primer is:
KF6-F：GATCCCGAAGGCCAACACAATAGG；
PLD-R：TAAGGTAATCCCTCGGGTCAG；
(5) plasmid molecule of new recombinant dna fragment clone.
Described molecular cloning refers to restructuring DNA fragment specific obtained above is connected on plasmid vector pMD18-T, obtains transgenosis standard positive plasmid molecule pMD-KTK, and the external source complete sequence of the plasmid molecule of acquisition is seen Fig. 2.
The quantitative and qualitative analysis PCR checking of the restructuring standard plasmid that (6) builds.
Described qualitative PCR checking, can amplify paddy rice internal standard gene PLD distinguished sequence and transgenic paddy rice KF6, TT51, KMD1 specificity of transformant sequence separately in the restructuring standard plasmid pMD-KTK that refers to build with the qualitative PCR method validation, and in pMD-KTK, the detection sensitivity of each specific sequence can satisfy the requirement that qualitative PCR detects.
described quantitative PCR checking, paddy rice internal standard gene PLD and transgenic paddy rice KF6 in the restructuring standard plasmid pMD-KTK that refers to build with the quantifying PCR method analysis, TT51, the limit of detection of KMD1 specificity of transformant sequence separately, quantitation limit and repeatability, the characteristics such as repeatability, to identify that this restructuring standard plasmid substitutes transgenic paddy rice KF6, TT51, the ability of KMD1 matrix positive criteria product, and utilize restructuring standard plasmid pMD-KTK to carry out quantitative analysis to the transgenic paddy rice sample of known content, and then verify that this restructuring standard plasmid is at transgenic paddy rice KF6, TT51, validity in the actual detection of KMD1 sample.
The invention still further relates to the application of described restructuring standard plasmid in the quantitative and qualitative analysis PCR of transgenic paddy rice KF6, TT51, KMD1 detects.
The invention still further relates to the test kit that a kind of qualitative PCR for transgenic paddy rice KF6, TT51, KMD1 detects, mainly comprise described restructuring standard plasmid and Auele Specific Primer; Described specific primer sequence is as follows:
KF6 flanking sequence Auele Specific Primer:
KF6-F：GATCCCGAAGGCCAACACAATAGG；
KF6-R：TCGTCCGAGGGCAAAGAAATAGAG；
TT51 flanking sequence Auele Specific Primer:
TT51-F：CCGGCGTCAATACGGGATAAT；
TT51-R：AGCCTCGTTGGATTTCTTACA；
KMD1 flanking sequence Auele Specific Primer:
KM-F：TGTGGAGCGTCAACTGGGAGC；
KM-R：GCTGTTGCCCGTCTCACTGGT；
The PLD gene-specific primer:
PLD-F：AGACCAGAAGACATTGAGCCG；
PLD-R：TAAGGTAATCCCTCGGGTCAG。
The invention still further relates to a kind of test kit of the quantitative PCR detection for transgenic paddy rice KF6, TT51, KMD1, mainly comprise described restructuring standard plasmid and Auele Specific Primer and probe; Described Auele Specific Primer and probe sequence are as follows:
KF6 flanking sequence Auele Specific Primer and probe:
KF6-F：GATCCCGAAGGCCAACACAATAGG；
KF6-R：TCGTCCGAGGGCAAAGAAATAGAG；
KF6-107P:FAM-ACGACCCGGCCAGTTAAGGCCA-TAMRA；
TT51 flanking sequence Auele Specific Primer and probe:
TT51-F：CCGGCGTCAATACGGGATAAT；
TT51-R：AGCCTCGTTGGATTTCTTACA；
TT51-120P：FAM-ATCTGCCCCAGCACTCGTCCG-BHQ1；
KMD1 flanking sequence Auele Specific Primer and probe:
KM-F：TGTGGAGCGTCAACTGGGAGC；
KM-R：GCTGTTGCCCGTCTCACTGGT；
KM-P:FAM-CGTCAATTTGTTTACACCACAATATATCCCG-TAMRA；
PLD gene-specific primer and probe:
PLD-F：AGACCAGAAGACATTGAGCCG；
PLD-R：TAAGGTAATCCCTCGGGTCAG；
TM013:FAM-TGTTGTGCTGCCAATGTGGCCTG-BHQ1。
Be the to recombinate design of standard plasmid and Auele Specific Primer of the key of this test kit, other compositions in test kit, for example the PCR reaction reagent, can select by this area routine.The PCR reaction reagent comprises PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase etc., and wherein the PCR damping fluid can adopt commercial commodity, also can prepare voluntarily, for example with Tris-HCl, KCl, MgCl
2Prepare Deng being mixed in proportion, when detecting, PCR reaction reagent and amplimer are mixed, add again testing sample or standard substance, can carry out pcr amplification reaction, pcr amplification product runs glue with 2% agarose, if the 250bp band appears in the pcr amplification product of sample, the expression testing sample contains paddy rice composition (otherwise not containing the paddy rice composition); Further, if the band of 250bp and 454bp occurs simultaneously, can judge and contain transgenic paddy rice KF6 composition; If the band of 250bp and 305bp occurs simultaneously, can judge and contain transgenic paddy rice TT51 composition; If the band of 250bp and 398bp occurs simultaneously, can judge and contain transgenic paddy rice KMD1 composition.
For reaching the requirement of detection by quantitative, can be at first take standard plasmid molecule pMD-KTK as calibration object, use Auele Specific Primer and TaqMan probe to carry out the real-time fluorescence quantitative PCR amplification, make the typical curve of specificity of transformant separately of paddy rice internal standard gene PLD typical curve and transgenic paddy rice KF6, TT51, KMD1; Extract the genomic dna of detected sample, as template, carry out the real-time fluorescence quantitative PCR amplification, the substitution typical curve, calculate the initial copy number of sample, then according to the copy number of the transformant event of transgenic paddy rice and the ratio of the copy number of paddy rice internal standard gene, can obtain the percentage composition of transgenic paddy rice KF6/TT51/KMD1 in sample.
The restructuring standard plasmid pMD-KTK that the present invention utilizes overlapping PCR and molecule clone technology to build first to contain paddy rice internal standard gene PLD and contain simultaneously the sequence of specificity of transformant separately of three transgenic paddy rice KF6, TT51, KMD1.The present invention's standard plasmid of recombinating can substitute the positive criteria product of transgenic paddy rice KF6, TT51 and KMD1, be used for transgenic paddy rice KF6, TT51, KMD1 specificity of transformant quantitative and qualitative analysis PCR are detected, solved well the problem of transgenic paddy rice KF6, TT51, KMD1 positive standard substance scarcity in testing process.This restructuring standard plasmid has the advantages such as specificity is good, sensitivity is high in reality detects, when using this restructuring standard plasmid and carrying out the actual sample quantitative analysis, in the allowed band that the deviation of sample detection result is extensively approved in the world.Therefore, the restructuring standard plasmid pMD-KTK of the present invention's structure is applicable to the quantitative and qualitative analysis PCR of transformant composition in transgenic paddy rice KF6, TT51, KMD1 and products thereof is detected fully.
(4) description of drawings
Fig. 1 is the schematic diagram of the standard plasmid molecule pMD-KTK that builds of the present invention;
Fig. 2 is whole exogenous arrays of the standard plasmid molecule pMD-KTK that builds of the present invention, and wherein KF6, TT51, KMD1 and PLD represent respectively transgenic paddy rice KF6, TT51, KMD1 specificity of transformant sequence separately and the distinguished sequence of paddy rice internal standard gene; The dash area sequence is the lap in overlapping PCR primer; The overlapping PCR primer of solid line tip Sequence and qualitative PCR primer; Dotted line tip sequence is quantification PCR primer, and solid line is probe without the tip sequence;
Fig. 3 is amplification curve and the typical curve of the fluorescent quantitation of each element of standard plasmid of building in the present invention.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the structure of restructuring standard plasmid pMD-KTK
One, experiment material
Transgenic paddy rice KF6, TT51 and KMD1.
Two, experiment reagent
PMD18-T carrier, ExTaq archaeal dna polymerase and damping fluid thereof, dNTPs, Marker are available from precious biotechnology (Dalian) company limited; Rice genome extraction test kit and quantitative PCR kit are available from Shanghai handsome biotechnology Services Co., Ltd; The PCR product reclaims test kit, plasmid extraction kit, DH5 α competence bacterial strain available from the Beijing Quanshijin Biotechnology Co., Ltd; Biochemical reagents are available from Shanghai living work Bioisystech Co., Ltd; Primer is synthetic by Shanghai handsome biotechnology Services Co., Ltd.
Three, key instrument equipment
PCR instrument (German Biometra), gel imaging system (U.S. GE), Ultrospec1100pro ultraviolet spectrophotometer (U.S. GE), other instruments comprise: thermostat water bath, whizzer, constant incubator, electronic balance, micropipet etc.
Four, experimental technique and process
1, overlapping PCR design of primers
Utilize the inquiry of GenBank and data of literatures to obtain the sequence of specificity of transformant separately of paddy rice internal standard gene PLD, transgenic paddy rice KF6, TT51 and KMD1; Utilize Primer Premier5 software, design four pairs of overlapping PCR primers, be respectively used to amplifying rice internal standard gene PLD, transgenic paddy rice KF6, TT51 and KMD1 specificity of transformant sequence.Primer sequence sees Table 1.
Table 1: the overlapping PCR primer that builds standard plasmid molecule pMD-KTK
2, the amplification of transgenic paddy rice specificity of transformant sequence and paddy rice internal standard gene PLD
Utilize the corresponding transgenic paddy rice of primer pair in table 1 to increase, the PCR system is: 10 * PCR damping fluid (Mg2+Plus), 200mmol dNTPs, 0.5mmol forward primer, 0.5mmol reverse primer, Taq archaeal dna polymerase and the DNA profiling of 1.25U.
The PCR program is 95 ℃ of 5min denaturations; 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 7min.
The PCR product runs glue and takes pictures with 2% agarose.Obtain the specificity of transformant sequence of transgenic paddy rice KF6, TT51, KMD1 and the distinguished sequence (SEQ ID No.1～4) of paddy rice internal standard gene PLD.
3, utilize overlapping PCR that four fragments of above-mentioned acquisition are spliced
The amplified production of KF6 and TT51 is got respectively 1 μ L add in reaction system as amplification template, amplification condition as above utilizes primer pair KF6-F/TT51-R-add to carry out overlapping pcr amplification, with KF6 and two fragments of TT51 link together (KF6+TT51).
The amplified production of KMD1 and PLD is got respectively 1 μ L add in reaction system as amplification template, amplification condition as above utilizes primer pair KM-F-add/PLD-R to carry out overlapping pcr amplification, with KMD1 and two fragments of PLD link together (KMD1+PLD).
The amplified production of above-mentioned twice overlapping PCR is got respectively 1 μ L to add in reaction system as amplification template, amplification condition as above, utilize primer pair KF6-F/PLD-R to carry out overlapping pcr amplification, two fragments that above-mentioned overlapping PCR is obtained link together (KF6+TT51+KMD1+PLD).The junction fragment that obtains is reclaimed order-checking, and result shows that the sequence (SEQ ID No.5) of acquisition is consistent with the amplified fragments of design.
4, recombinant fragment clone
Utilize molecular cloning method that the recombinant fragment that above-mentioned overlapping pcr amplification obtains is connected on plasmid vector pMD18-T, linked system sees Table 2.To connect product and transform DH5 α competence bacterial strain.Utilize bacterium colony PCR that recombinant bacterial strain is identified, extract plasmid pMD-KTK according to the plasmid extraction kit specification sheets.Determine that by sequencing analysis the exogenous array in the restructuring standard plasmid is consistent with expection.
Table 2: linked system
Embodiment 2: the application of restructuring standard plasmid pM D-KTK in reality detects
One, experiment material
Transgenic paddy rice KF6, TT51, KMD1; Genetically engineered soybean GTS40-3-2, MON89788; Transgenic corns MON810, Bt11, Bt176; Transgene cotton MON15985, MON88913, transgene rape MS1, GT73; And other crops such as non-transgenic paddy rice, soybean, corn and cotton.
Two, reagent
Taq archaeal dna polymerase and damping fluid thereof, dNTPs, Marker are available from precious biotechnology (Dalian) company limited; Plasmid extraction kit is available from the Beijing Quanshijin Biotechnology Co., Ltd; 2 * Mix TaqMan quantitative PCR kit is available from Shanghai handsome biotechnology Services Co., Ltd; TaqMan probe and primer are synthetic by Shanghai handsome biotechnology Services Co., Ltd.
Three, key instrument equipment
7500 type fluorescent PCR instrument (American AB I), PCR instrument (German Biometra), gel imaging system (U.S. GE), Ultrospec1100pro ultraviolet spectrophotometer (U.S. GE), other instruments comprise: thermostat water bath, whizzer, constant incubator, electronic balance, micropipet etc.
Four, experimental technique and process
1, plant genome DNA extracts
Get appropriate plant sample, fully grind in liquid nitrogen, take the ground sample of 100mg (that need not grind can directly take), extract according to Plant Genome the genomic dna that the test kit specification sheets extracts plant sample.The quality of DNA sample is estimated with the method that agarose gel electrophoresis and nucleic acid quantification detect.
2, extraction and the detection of restructuring standard plasmid pMD-KTK
To contain the intestinal bacteria of standard plasmid molecule pMD-KTK in 37 ℃ of cultivation 10h, extract plasmid DNA according to the plasmid extraction kit specification sheets.Get 3 μ L DNA samples, detect with 1% agarose gel electrophoresis, judgement DNA integrity; Detect DNA purity and concentration with spectrophotometer, calculate OD
260nm/ OD
280nmRatio in 1.8 left and right, meet qualitative, quantitative PCR detection requirement; According to restructuring standard plasmid DNA starting point concentration and size, with 0.1 * TE damping fluid, it is diluted to 2.6 * 10 successively
5, 2.6 * 10
4, 2.6 * 10
3, 2.6 * 10
2, 2.6 * 10,2.6 and 0.26copy μ L
-1, 4 ℃ save backup.
3, the application of restructuring standard plasmid pMD-KTK in qualitative PCR detects
According to the specificity of transformant sequence of paddy rice internal standard gene PLD and transgenic paddy rice KF6, TT51 and KMD1, use primer to carry out qualitative PCR, the examination criteria plasmid molecule detects effect.The primer sequence that uses sees Table 3.
Table 3: restructuring standard plasmid qualitative PCR detects primer
Utilize the primer in table 3 to increase, the PCR system is: 10 * PCR damping fluid (Mg2+Plus), 200mmol dNTPs, 0.5mmol forward primer, 0.5mmol reverse primer, Taq archaeal dna polymerase and the DNA profiling of 1.25U.
The PCR program is 95 ℃ of 5min denaturations; 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 7min.The PCR product runs glue and takes pictures with 2% agarose.
With restructuring standard plasmid pMD-KTK(2.6 * 10
3Copy μ L
-1), the DNA of transgenic paddy rice KF6, TT51 and KMD1, other transgenic lines and non-transgenic crop is that template is carried out qualitative PCR and detected, judgement restructuring standard plasmid is used for the specificity that qualitative PCR detects, result only shows that recombinant plasmid just has amplification in corresponding transformation event, shows that recombinant plasmid has good specificity.
DNA sample (2.6 * 10 with the restructuring standard plasmid pMD-KTK of different concns
5, 2.6 * 10
4, 2.6 * 10
3, 2.6 * 10
2, 2.6 * 10,2.6 and 0.26copy μ L
-1) carrying out the qualitative PCR amplification for template, judgement restructuring standard plasmid is used for the sensitivity that qualitative PCR detects, and result shows that the sensitivity that qualitative PCR detects can reach 13 copies.
4, standard plasmid molecule pMD-KTK is used for the transgenosis detection by quantitative
Auele Specific Primer and TaqMan that the quantitative PCR detection of transgenic paddy rice KF6, TT51 suitable according to the data of literatures design and KMD1 specificity of transformant sequence and paddy rice internal standard gene PLD is used, the sequence of probe primer and probe sees Table 4.
Table 4: restructuring standard plasmid quantitative PCR detection primer
Annotate:
aProbe 5 ' end and 3 ' end be mark reporter group FAM and quenching group BHQ1 respectively;
bProbe 5 ' end and 3 ' end be mark reporter group FAM and quenching group TAMRA respectively.
The quantitative PCR reaction volume is 25 μ L, comprising: the 12.5 general Mix of μ L2 * TaqMan, DNA profiling (5 μ L), each 300nmol/L of primer, the water of 100nmol/L probe and 5.2 μ L.
The reaction conditions of quantitative pcr amplification is: 95 ℃ of 10min; 95 ℃ of 10s, 60 ℃ of 60s, totally 45 circulations.Parallel 3 reactions, the repeated experiments 3 times done of each sample.
The limit of detection of restructuring standard plasmid quantitative PCR and the foundation of quantitation limit and typical curve thereof.Process is carried out quantitative pcr amplification to the DNA sample of the restructuring standard plasmid pMD-KTK of different concns, in order to improve the detected result accuracy, the quantitative PCR detection LOD of the specificity of transformant sequence of the paddy rice internal standard PLD gene of restructuring standard plasmid pMD-KTK and transgenic paddy rice KF6, TT51, KMD1 is decided to be 13 copies, LOQ is 130 copies, has good repeatability and repeatability.
Take concentration as 2.6 * 10
5, 2.6 * 10
4, 2.6 * 10
3, 2.6 * 10
2, 2.6 * 10 restructuring standard plasmid pMD-KTK is that standard substance are set up PLD gene and transgenic paddy rice KF6, TT51, KMD1 specificity of transformant sequence quantitative PCR typical curve (see figure 3) separately, the facies relationship number average of four typical curves is greater than 0.98, slope is between-3.1～-3.6, has good linear relationship, the alternative transgenic paddy rice KF6 of restructuring standard plasmid pMD-KTK of the present invention's structure, the positive criteria product of TT51, KMD1 are described, satisfy the quantitative PCR detection that is used for transgenic paddy rice KF6, TT51, KMD1 and products thereof.
The PLD gene that utilization restructuring standard plasmid pMD-KTK sets up and transgenic paddy rice KF6, TT51, KMD1 specificity of transformant sequence quantitative PCR typical curve separately, to containing respectively transgenic paddy rice KF6, TT51, KMD1(3%, 0.5%) sample carry out quantitative analysis, quantitative analysis results is as shown in table 5, the deviation of detected result and actual value is between 8.64%～22%, and standard deviation is between 0.03～0.11.Show that utilization restructuring standard plasmid pMD-KTK is more accurate to the result of actual sample analysis, in 0～25% scope that the deviation of detected result is extensively approved in the world, show the alternative corresponding transgenic paddy rice positive criteria product of restructuring standard plasmid pMD-KTK that the present invention builds, be applicable to transgenic paddy rice KF6, TT51, KMD1 specificity of transformant quantitative PCR analysis and detection.
Table 5: transgenosis KF6, TT51, KMD1 content separately in the detection by quantitative sample
Above result shows, the standard plasmid pMD-KTK that the present invention builds is fit to the quantitative and qualitative analysis PCR of transgenic paddy rice KF6, TT51, KMD1 is detected, for the control of transgenic product provides technical support.
Claims (5)
1. restructuring standard plasmid that the quantitative and qualitative analysis PCR that is used for transgenic paddy rice KF6, TT51, KMD1 detects, obtain by the following method: the PLD gene that the TT51 flanking sequence shown in the KF6 flanking sequence shown in SEQ ID No.1, SEQ ID No.2, the KMD1 flanking sequence shown in SEQ ID No.3 are connected with SEQ ID No.4 connects acquisition fusion fragment successively, gained being merged fragment is cloned in carrier pMD18-T again, obtain described restructuring standard plasmid, be designated as pMD-KTK.
2. restructuring standard plasmid as claimed in claim 1, is characterized in that described fusion fragment nucleotide sequence is as shown in SEQ ID No.5.
3. the application of restructuring standard plasmid as claimed in claim 1 in the quantitative and qualitative analysis PCR of transgenic paddy rice KF6, TT51, KMD1 detects.
4. a test kit that is used for the qualitative PCR detection of transgenic paddy rice KF6, TT51, KMD1, mainly comprise restructuring standard plasmid claimed in claim 1 and Auele Specific Primer; Described specific primer sequence is as follows:
KF6 flanking sequence Auele Specific Primer:
KF6-F：GATCCCGAAGGCCAACACAATAGG；
KF6-R：TCGTCCGAGGGCAAAGAAATAGAG；
TT51 flanking sequence Auele Specific Primer:
TT51-F：CCGGCGTCAATACGGGATAAT；
TT51-R：AGCCTCGTTGGATTTCTTACA；
KMD1 flanking sequence Auele Specific Primer:
KM-F：TGTGGAGCGTCAACTGGGAGC；
KM-R：GCTGTTGCCCGTCTCACTGGT；
The PLD gene-specific primer:
PLD-F：AGACCAGAAGACATTGAGCCG；
PLD-R：TAAGGTAATCCCTCGGGTCAG。
5. a test kit that is used for the quantitative PCR detection of transgenic paddy rice KF6, TT51, KMD1, mainly comprise restructuring standard plasmid claimed in claim 1 and Auele Specific Primer and probe; Described Auele Specific Primer and probe sequence are as follows:
KF6 flanking sequence Auele Specific Primer and probe:
KF6-F：GATCCCGAAGGCCAACACAATAGG；
KF6-R：TCGTCCGAGGGCAAAGAAATAGAG；
KF6-107P:FAM-ACGACCCGGCCAGTTAAGGCCA-TAMRA；
TT51 flanking sequence Auele Specific Primer and probe:
TT51-F：CCGGCGTCAATACGGGATAAT；
TT51-R：AGCCTCGTTGGATTTCTTACA；
TT51-120P：FAM-ATCTGCCCCAGCACTCGTCCG-BHQ1；
KMD1 flanking sequence Auele Specific Primer and probe:
KM-F：TGTGGAGCGTCAACTGGGAGC；
KM-R：GCTGTTGCCCGTCTCACTGGT；
KM-P:FAM-CGTCAATTTGTTTACACCACAATATATCCCG-TAMRA；
PLD gene-specific primer and probe:
PLD-F：AGACCAGAAGACATTGAGCCG；
PLD-R：TAAGGTAATCCCTCGGGTCAG；
TM013:FAM-TGTTGTGCTGCCAATGTGGCCTG-BHQ1。
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| CN107312817A (en) * | 2016-04-25 | 2017-11-03 | 中国检验检疫科学研究院 | Primed probe, kit and the method precisely quantitatively detected for genetically modified rice Kemingdao strain-specific gene composition |
| CN106480216A (en) * | 2016-12-07 | 2017-03-08 | 江苏省农业科学院 | A kind of standard plasmid for detecting transgenic regulation element and its construction method and application |
| CN110257542A (en) * | 2019-06-12 | 2019-09-20 | 中国检验检疫科学研究院 | Detect DNA standard items and its application of rich No. 6 of transgenic paddy rice section |
| CN110724704A (en) * | 2019-10-16 | 2020-01-24 | 安徽省农业科学院水稻研究所 | Positive plasmid for identifying transgenic rice transformant and construction method and application thereof |
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