CN104032007A - Method for detecting exogenous gene copy number in transgenic tobacco - Google Patents
Method for detecting exogenous gene copy number in transgenic tobacco Download PDFInfo
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Abstract
The invention discloses a method for detecting an exogenous gene copy number in transgenic tobacco. A real-time fluorescent quantitation PCR technology is utilized to simply, rapidly and accurately detect the exogenous gene copy number in transgenic tobacco. The invention discloses a pair of primers consisting of DNA molecules shown in (1) and (2), namely (1) a DNA molecule shown in SEQ ID No.3; and (2) a DNA molecule shown in SEQ ID No.4. A single-copy endogenous gene ribonucleotide reductase gene (RNR2) of the tobacco is selected as a reference gene, and the exogenous gene copy number in transgenic tobacco is detected through a SYBR GreenI real-time fluorescent quantitation PCR technology. The method disclosed by the invention provides a new thought for copy number analysis of the transgenic tobacco, and the screening requirement for excellent transformation plants in genetic engineering is met.
Description
Technical field
The present invention relates to a kind of method that detects copy number of foreign gene in transgene tobacco; Be particularly related to a kind of method of utilizing Real-Time Fluorescent Quantitative PCR Technique to detect copy number of foreign gene in transgene tobacco, belong to biological technical field.
Background technology
In recent years, plant transgenic technology is widely used in and produces pharmaceutical protein, improves crop quality, improves the research in the fields such as crop yield.The method that plant transgene adopts is at present mainly agrobacterium tumefaciens mediated method, but the T-DNA region of containing goal gene that Agrobacterium is entrained is in random insertion recipient plant genome, thereby the transfer-gen plant obtaining often contains the foreign gene of one or more copies.In transgenic plant, the copy number of foreign gene is the key factor that affects the expression amount of this gene, and the foreign gene of multiple copied can directly cause gene silencing or can not stably express.Tobacco (Nicotiana tabacum) is a kind of plant of dicots Solanaceae, at present widely as the model plant of genetically engineered research, and the copy number of foreign gene is the key factor that directly affects expression level and the genetic stability of goal gene in transgene tobacco, therefore, identifying the copy number of foreign gene in transgene tobacco is the basis of carrying out follow-up study.
At present, in the research in genetic transformation later stage of plant, determine whether foreign gene is incorporated on transformed plant genome, and the copy number of definite transformed plant is very important for the research in later stage, traditional method is mainly to use Southern hybridization, the accuracy of the method has obtained general approval, but the method wastes time and energy, each swimming lane needs the shortcomings such as DNA sample of 10-30 μ g, if foreign gene is recombinated when inserting conversion of plant genome, cause the loss of restriction enzyme site, just can not carry out the detection of copy number with Southern hybridization, the method needs expensive reagent, the time of cost is more, the DNA material needing is also more, and the resistance seedling that plant tissue culture is differentiated to form is often unsuitable for a large amount of samplings a little less than, also there is the shortcomings such as the multiple copied insertion point identifying is inaccurate.Real time fluorescence quantifying PCR method is in PCR reaction system, to add nonspecific fluorescence dye or specific fluorescent probe, and the variation of fluorescence volume in Real-Time Monitoring reaction system obtains and reaches the required cycle number Ct of certain fluorescence volume (threshold value).Standard substance and corresponding Ct value production standard curve thereof by concentration known, just can carry out quantitatively unknown sample accurately.Than Southern hybridization, the method DNA amount convenient, fast, that need is less and do not need to use radioactive substance, and due to the high specificity of the method, thereby the experimental result obtaining is more accurately true.Real-time fluorescence quantitative PCR not only can increase to specific T-DNA sequence, also can detect gene recombination in transgenic strain.Because the growth cycle of plant is longer, real time fluorescent quantitative method is used less material just can detect, and therefore at the early stage of plant culturing, just can detect screening, has avoided the work of a large amount of groups of trainings.Thereby Real-Time Fluorescent Quantitative PCR Technique is a kind of method that feasible transfer-gen plant copy number is analyzed.
In research in the past, find, the copy number results of real-time fluorescence quantitative PCR detection and Southern hybridization gained is very approaching, but has partial results to show a little higher than Southern hybridization of copy number of real-time fluorescence quantitative PCR gained.During the copy number of the foreign gene in detecting transgenic corns such as Ingham, find, the copy number results of real-time fluorescence quantitative PCR gained is hybridized higher than Southern.This situation may cause due to multiple factors, such as enzyme in Southern hybridization cut digestion not exclusively, the factors such as height of gene rearrangement, development background all may cause the result of this mistake.Thereby the copy number results that real-time fluorescence quantitative PCR obtains is more close to objective fact.
Develop a kind of real time fluorescence quantifying PCR method identify copy number of foreign gene in transgene tobacco that can detect and there is material impact to carrying out follow-up study.
Summary of the invention
The object of this invention is to provide a kind of method that detects copy number of foreign gene in transgene tobacco, what utilize is Real-Time Fluorescent Quantitative PCR Technique, utilizes method provided by the invention can detect simply, fast and accurately copy number of foreign gene in transgene tobacco.
The invention provides pair of primers, by the DNA molecular shown in following (1) and (2), formed:
(1) DNA molecular shown in SEQ ID No.3;
(2) DNA molecular shown in SEQ ID No.4.
The test kit that detects copy number of foreign gene in transgene tobacco also belongs to a protection scope of the present invention, and this test kit comprises above-mentioned primer.
Described test kit also comprises working instructions, in specification sheets, record and the contents are as follows: the genomic dna of non-transgenic tobacco of gradient dilution of take is template, with the DNA molecular shown in the DNA molecular shown in SEQ ID No.3 and SEQ ID No.4, be respectively upstream primer and downstream primer, carry out real-time fluorescence quantitative PCR amplification; The concentration of the genomic dna template of the non-transgenic tobacco of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of genomic dna template of other dilution non-transgenic tobaccos and the genomic dna template of the non-transgenic tobacco of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating; The lg value of copy number concentration value of take is ordinate zou, and corresponding Ct value be X-coordinate, and drafting reference gene typical curve, obtains reference gene typical curve formula 1, and the while take the genomic dna of transgene tobacco to be detected and carries out above-mentioned experiment as template, obtains Ct value X
1; By Ct value X
1bring reference gene typical curve formula 1 into, obtain the lg value of the copy number concentration value of the reference gene in transgene tobacco to be detected, i.e. Y
1;
The plasmid that contains foreign gene of gradient dilution of take is template, and upstream primer and the downstream primer of foreign gene of take is primer, carries out real-time fluorescence quantitative PCR amplification; The concentration of the plasmid template that contains foreign gene of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of other dilution plasmid templates that contain foreign gene and the plasmid template that contains foreign gene of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating; The lg value of copy number concentration value of take is ordinate zou, and corresponding Ct value, for X-coordinate drafting foreign gene typical curve, obtains foreign gene typical curve formula 2, and the while take the genomic dna of transgene tobacco to be detected and carries out above-mentioned experiment as template, obtains Ct value X
2; By Ct value X
2bring foreign gene typical curve formula 2 into, obtain the lg value of the copy number concentration value of the foreign gene in transgene tobacco to be detected, i.e. Y
2;
Y
2/ Y
1be the copy number of the foreign gene in transgene tobacco to be detected, work as Y
2/ Y
1after radix point, first is greater than at 5 o'clock, and integral part adds 1, is less than or equal at 5 o'clock, and integral part is constant, and integral part is the copy number of foreign gene in transgene tobacco;
Described extent of dilution minimum refers to that extension rate is minimum;
The concentration unit of described template is identical;
Described reference gene is ribonucleotide reductase (Ribonucleotide reductase) RNR2;
The concentration of the genomic dna of the non-transgenic tobacco of described gradient dilution is respectively 0.0406 μ g/ μ L, 0.00812 μ g/ μ L, 0.001624 μ g/ μ L, 0.0003248 μ g/ μ L, 0.00006496 μ g/ μ L;
Described non-transgenic tobacco is specially non-transgenic tobacco bred K346;
The reaction system of described real-time fluorescence quantitative PCR is: template 2 μ L, 10 μ molL
-1each 0.3 μ L of upstream primer, downstream primer,
premix Ex Taq
tMiI10 μ L, adds ddH
2o supplies system to 20 μ L;
Described
premix Ex Taq
tMiI is specifically purchased from precious biotechnology (Dalian) company limited;
The reaction system of described real-time fluorescence quantitative PCR is: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 30s, 40 circulations.
A kind of method that detects copy number of foreign gene in transgene tobacco also belongs to protection scope of the present invention, the genomic dna of non-transgenic tobacco of gradient dilution of take is template, with the DNA molecular shown in SEQ ID No.3 and the DNA molecular shown in SEQ IDNo.4, be respectively upstream primer and downstream primer, carry out real-time fluorescence quantitative PCR amplification; The concentration of the genomic dna template of the non-transgenic tobacco of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of genomic dna template of other dilution non-transgenic tobaccos and the genomic dna template of the non-transgenic tobacco of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating; The lg value of copy number concentration value of take is ordinate zou, and corresponding Ct value be X-coordinate, and drafting reference gene typical curve, obtains reference gene typical curve formula 1, and the while take the genomic dna of transgene tobacco to be detected and carries out above-mentioned experiment as template, obtains Ct value X
1; By Ct value X
1bring reference gene typical curve formula 1 into, obtain the lg value of the copy number concentration value of the reference gene in transgene tobacco to be detected, i.e. Y
1;
The plasmid that contains foreign gene of gradient dilution of take is template, and upstream primer and the downstream primer of foreign gene of take is primer, carries out real-time fluorescence quantitative PCR amplification; The concentration of the plasmid template that contains foreign gene of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of other dilution plasmid templates that contain foreign gene and the plasmid template that contains foreign gene of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating; The lg value of copy number concentration value of take is ordinate zou, and corresponding Ct value, for X-coordinate drafting foreign gene typical curve, obtains foreign gene typical curve formula 2, and the while take the genomic dna of transgene tobacco to be detected and carries out above-mentioned experiment as template, obtains Ct value X
2; By Ct value X
2bring foreign gene typical curve formula 2 into, obtain the lg value of the copy number concentration value of the foreign gene in transgene tobacco to be detected, i.e. Y
2;
Y
2/ Y
1be the copy number of the foreign gene in transgene tobacco to be detected, work as Y
2/ Y
1after radix point, first is greater than at 5 o'clock, and integral part adds 1, is less than or equal at 5 o'clock, and integral part is constant, and integral part is the copy number of foreign gene in transgene tobacco;
The concentration unit of described template is identical;
Described reference gene is ribonucleotide reductase gene RNR2;
Described foreign gene is the gene proceeding in transgene tobacco.
In aforesaid method, the concentration of the genomic dna of the non-transgenic tobacco of described gradient dilution is respectively 0.0406 μ g/ μ L, 0.00812 μ g/ μ L, 0.001624 μ g/ μ L, 0.0003248 μ g/ μ L, 0.00006496 μ g/ μ L.
In above-mentioned arbitrary described method, the reaction system of described real-time fluorescence quantitative PCR is: template 2 μ L, 10 μ molL
-1each 0.3 μ L of upstream primer, downstream primer,
premix Ex Taq
tMiI10 μ L, adds ddH
2o supplies system to 20 μ L;
premix Ex Taq
tMiI is specifically purchased from precious biotechnology (Dalian) company limited;
The reaction system of described real-time fluorescence quantitative PCR is: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 30s, 40 circulations.
Described reference gene typical curve formula 1 is specially Y
1=-0.2858X
1+ 5.6695.
In above-mentioned arbitrary described method, described non-transgenic tobacco is non-transgenic tobacco bred K346.
The foreign gene of described transgene tobacco is specially GFP gene;
Specifically by importing by agriculture bacillus mediated leaf dish method method non-transgenic tobacco, pBI121-GFP plasmid obtains described transgene tobacco;
Described non-transgenic tobacco is specially non-transgenic tobacco bred K346;
The concentration of the plasmid that contains foreign gene of described gradient dilution is specifically respectively 0.0356 μ g/ μ L, 0.00712 μ g/ μ L, 0.001424 μ g/ μ L, 0.0002848 μ g/ μ L, 0.00005696 μ g/ μ L;
The upstream primer of described foreign gene and downstream primer are specially respectively the DNA molecular shown in the DNA molecular shown in SEQ ID No.5 and SEQ ID No.6;
Described foreign gene typical curve formula 2 is specially Y
2=-0.2826X
2+ 2.1048.
The application in copy number of foreign gene in detecting transgene tobacco of above-mentioned primer, mentioned reagent box also belongs to protection scope of the present invention.
The foreign gene of described transgene tobacco is specially GFP gene;
Specifically by importing by agriculture bacillus mediated leaf dish method method non-transgenic tobacco, pBI121-GFP plasmid obtains described transgene tobacco;
Described non-transgenic tobacco is specially non-transgenic tobacco bred K346.
The present invention selects the native gene ribonucleotide reductase gene (RNR2) of the single copy of tobacco self as reference gene, detects the copy number of foreign gene in transgene tobacco by Real-Time Fluorescent Quantitative PCR Technique.Method provided by the invention can provide a kind of new thinking for the copy number analysis of transgene tobacco from now on, meets the screening requirements to good transformed plant in genetically engineered.
Accompanying drawing explanation
Fig. 1 is that the PCR that turns GFP genetic tobacco detects.
Fig. 2 is reference gene RNR2 typical curve.
Fig. 3 is foreign gene GFP typical curve.
Fig. 4 is reference gene RNR2 melting curve.
Fig. 5 is foreign gene GFP melting curve.
Fig. 6 is the fail-safe analysis of reference gene RNR2 primer.
Fig. 7 is the fail-safe analysis of foreign gene GFP primer.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Non-transgenic tobacco bred K346 (Nicotiana tabacum cv.346) document " Luo Cong. mango genetic diversity and adverse circumstance and important flowering time genes involved research [doctorate paper]. Nanning: Guangxi University; 2012 " in disclose, the acquisition of public Ke Cong Veterinary Institute of Guangxi Zhuang Autonomous Region.
PBI121-GFP plasmid document " Luo Cong. mango genetic diversity and adverse circumstance and important flowering time genes involved research [doctorate paper]. Nanning: Guangxi University, 2012 " in disclose, the acquisition of public Ke Cong Veterinary Institute of Guangxi Zhuang Autonomous Region.
The building process of following embodiment transfer GFP genetic tobacco is as follows: the plant expression vector pBI121-GFP that contains foreign gene GFP is proceeded to non-transgenic tobacco bred K346 by agriculture bacillus mediated leaf dish method, by following PCR screening, obtain the positive tobacco that turns GFP gene.
1, get the young leaflet tablet that agriculture bacillus mediated leaf dish method is screened the transformation of tobacco plant obtaining, with improved method of CTAB extract tobacco gene group DNA (method reference literature " and Luo Cong. mango genetic diversity and adverse circumstance and important flowering time genes involved research [doctorate paper]. Nanning: Guangxi University, 2012 ").2, according to the upper GFP gene order design of plant expression vector pBI121-GFP special primer, primer sequence is GFP1:5 '-ATGGGTAAAGGAGAAGAACT-3 ' (SEQ ID No.1), GFP2:5 '-TTATTTGTATAGTTCATCCA-3 ' (SEQ ID No.2) (reference literature " Luo Cong. mango genetic diversity and adverse circumstance and important flowering time genes involved research [doctorate paper]. Nanning: Guangxi University; 2012 "), object band is 718bp.3, take the genomic dna that step 1 obtains is template, take GFP1 and GFP2 carries out pcr amplification as primer, and using pBI121-GFP as template is as positive control, the genomic dna of non-transgenic tobacco K346 is the negative contrast of template, carry out above-mentioned PCR detection, obtain each pcr amplification product, the agarose gel electrophoresis of each product as shown in Figure 1.In Fig. 1,1 positive contrast; 2 negative contrasts; 3-12 is transfer-gen plant; M is DL2000Marker.The positive that screening acquisition has 718bp object band turns the tobacco of GFP gene as the material of the real-time fluorescence quantitative PCR detection copy number in following embodiment.
premix Ex Taq
tMiI is purchased from precious biotechnology (Dalian) company limited.
Embodiment 1, Real-Time Fluorescent Quantitative PCR Technique detect copy number of foreign gene in transgene tobacco
One, design of primers
Select the endogenous single copy gene-ribonucleotide reductase of tobacco (Ribonucleotide reductase) RNR2 (copy number of this gene disclosed in document " Chaboute M E; Combettes B; Clement B; et al.Molecular characterization of tobacco ribonucleotide reductase RNRI and RNR2cDNAs and cell cycle regulated expression in synchronized plant cells.Plant Molecular Biology; 1998,38:797-806 ") as reference gene.The primer of design reference gene RNR2 and foreign gene GFP is as follows:
Upstream primer RNR2U:5 '-TACCCGTCACTGGGAAAC-3 ' (SEQ ID No.3)
Downstream primer RNR2D:5 '-GGAAGCCGTAGAAAGCAC-3 ' (SEQ ID No.4)
Take RNR2U and the RNR2D reference gene RNR2 in primer amplification tobacco, object fragment length is 158bp;
Upstream primer GFPTU:5 '-AACTACTACTCCCACAACG-3 ' (SEQ ID No.5)
Downstream primer GFPTD:5 '-ATGGTCTGCTGGTGAACG-3 ' (SEQ ID No.6)
Take GFPTU and the GFPTD foreign gene GFP in primer amplification tobacco, object fragment length is 112bp.
Two, real-time fluorescence quantitative PCR detects copy number of foreign gene
(1) real-time fluorescence quantitative PCR reaction system and program
Real-time fluorescence quantitative PCR reaction system: template 2 μ L, 10 μ molL
-1each 0.3 μ L of upstream and downstream primer,
premix Ex Taq
tMiI10 μ L, adds ddH
2o complements to 20 μ L.
Real-time fluorescence quantitative PCR response procedures: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 30s, 40 circulations.After having reacted, carry out melting curve analysis.
Real-time fluorescence quantitative PCR reaction is carried out on Roche Light cycler2.0 quantitative real time PCR Instrument.
(2) making of reference gene typical curve
1, extract the genomic dna of non-transgenic tobacco bred K346 as the standard substance of reference gene RNR2, it is 0.0406 μ g/ μ L that ultraviolet spectrophotometry records genomic dna concentration, carries out 5 times of gradient dilutions, and extension rate is respectively 5
0, 5
-1, 5
-2, 5
-3, 5
-4, obtain the reference gene standard substance of 5 concentration dilutions.The concentration of the genomic dna of the non-transgenic tobacco of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of genomic dna of other dilution non-transgenic tobaccos and the genomic dna of the non-transgenic tobacco of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating, the copy number concentration of trying to achieve is respectively 5
0copy/μ L, 5
-1copy/μ L, 5
-2copy/μ L, 5
-3copy/μ L, 5
-4copy/μ L.Extract the genomic dna that 5 strains turn GFP genetic tobacco, as 5, turn GFP genetic tobacco and detect sample.
2, usining respectively the reference gene standard substance, 5 of 5 concentration dilutions turns GFP genetic tobacco and detects sample, water (blank) as template, the reference gene RNR2 primer (RNR2U and RNR2D) of take is primer, carry out the real-time fluorescence quantitative PCR amplification of step (), each sample arranges three repetitions.
The lg value of original template copy number concentration of reference gene standard substance of 5 concentration dilutions of take is ordinate zou, and corresponding Ct value is X-coordinate drawing standard curve, obtains typical curve formula, and result as shown in Figure 2.
(3) making of foreign gene typical curve
1, using pBI121-GFP as the standard substance of foreign gene GFP, concentration is 0.0356 μ g/ μ L, carries out 5 times of gradient dilutions, and extension rate is respectively 5
0, 5
-1, 5
-2, 5
-3, 5
-4, obtain the foreign gene standard substance of 5 concentration dilutions.The concentration of the plasmid that contains foreign gene of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of other dilution plasmids that contain foreign gene and the plasmid that contains foreign gene of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating; The copy number concentration of trying to achieve is respectively 5
0copy/μ L, 5
-1copy/μ L, 5
-2copy/μ L, 5
-3copy/μ L, 5
-4copy/μ L.
2, usining respectively the foreign gene standard substance, 5 of 5 concentration dilutions turns genomic dna that GFP genetic tobacco detects sample (detecting samples with 5 that are extracted in two), non-transgenic tobacco bred K346, water (blank) as template, the foreign gene GFP primer (GFPTU and GFPTD) of take is primer, carry out the real-time fluorescence quantitative PCR amplification of step (), each sample arranges three repetitions.
The lg value of original template copy number concentration of foreign gene standard substance of 5 concentration dilutions of take is ordinate zou, and corresponding Ct value is X-coordinate drawing standard curve, obtains typical curve formula, and result as shown in Figure 3.
Fig. 2 shows, the typical curve formula of reference gene RNR2 is Y
1=-0.2858X
1+ 5.6695, R
2=0.9994.
Fig. 3 shows, the typical curve formula of foreign gene GFP is Y
2=-0.2826X
2+ 2.1048, R
2=0.9989.
The relation conefficient of two standard lines formula, all close to 1, therefore can be carried out quantitatively sample accurately.
(4) primer specificity
The melting curve of RNR2 gene as shown in Figure 4.
The melting curve of GFP gene as shown in Figure 5.
From Fig. 4 and Fig. 5, can find out, the melting curve of RNR2 gene and GFP gene is all unimodal, illustrates that primer specificity is separately better, in reaction, without non-specific amplification, without primer dimer, illustrates that the real time fluorescent quantitative data that obtain are reliable.
(5) detection by quantitative of foreign gene in transgene tobacco
The reference gene standard substance, 5 of 5 concentration dilutions of usining in step (two) turn GFP genetic tobacco and detect sample, water (blank) as template, the reference gene RNR2 primer (RNR2U and RNR2D) of take is primer, carry out the real-time fluorescence quantitative PCR amplification of step () as shown in Figure 6, amplification shows that all tobacco plants all have amplification, and the not amplification of blank water.
The foreign gene standard substance, 5 of 5 concentration dilutions of usining respectively in step (three) turn genomic dna that GFP genetic tobacco detects sample, non-transgenic tobacco bred K346, water (blank) as template, the foreign gene GFP primer (GFPTU and GFPTD) of take is primer, carry out the real-time fluorescence quantitative PCR amplification of step () as shown in Figure 7, amplification shows that the sample that turns GFP genetic tobacco plant has amplification, but not amplification of transgene tobacco kind K346 sample and blank, interpret sample is not polluted, credible result.
(6) it is as shown in table 1 that 5 strains that obtained by step (two) turn Ct value and the Tm value of foreign gene GFP in these tobaccos that in GFP genetic tobacco, reference gene RNR2 and step (three) obtain.
Ct value and the Tm value of RNR2 and GFP gene in table 1 detection sample
(Tm in table 1 refers to the unwind temperature of a half of double chain DNA molecule, is for confirming the specific of PCR product)
The Ct value X of the RNR2 of GFP genetic tobacco detection sample gained will respectively be turned
1bring the reference gene typical curve formula that step (two) is made into, obtain the lg value of the template copy number concentration of reference gene RNR2, i.e. Y
1; The Ct value X of the GFP of GFP genetic tobacco detection sample gained will respectively be turned
2bring the foreign gene typical curve formula that step (three) is made into, obtain the lg value of the template copy number concentration of foreign gene GFP, i.e. Y
2; Y
2/ Y
1be the corresponding copy number that turns GFP genetic tobacco foreign gene GFP, after ratio radix point first integral part adds 1, is less than or equal at 5 o'clock while being greater than 5, integral part is constant, integral part is the copy number that turns GFP genetic tobacco foreign gene GFP.
Calculation result is as shown in table 2.Table 2 shows, 5 strains of detection turn GFP genetic tobacco, and copy number is 5 a strain, copy number is 8 have a strain, copy number be 19,28,45 respectively have a strain.
Table 2 transgene tobacco GFP gene copy number calculates
Claims (7)
1. pair of primers, is comprised of the DNA molecular shown in following (1) and (2):
(1) DNA molecular shown in SEQ ID No.3;
(2) DNA molecular shown in SEQ ID No.4.
2. detect a test kit for copy number of foreign gene in transgene tobacco, this test kit comprises primer claimed in claim 1.
3. a method that detects copy number of foreign gene in transgene tobacco, the genomic dna that comprises the steps: to take the non-transgenic tobacco of gradient dilution is template, with the DNA molecular shown in the DNA molecular shown in SEQ ID No.3 and SEQ ID No.4, be respectively upstream primer and downstream primer, carry out real-time fluorescence quantitative PCR amplification; The concentration of the genomic dna template of the non-transgenic tobacco of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of genomic dna template of other dilution non-transgenic tobaccos and the genomic dna template of the non-transgenic tobacco of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating; The lg value of copy number concentration value of take is ordinate zou, and corresponding Ct value be X-coordinate, and drafting reference gene typical curve, obtains reference gene typical curve formula 1, and the while take the genomic dna of transgene tobacco to be detected and carries out above-mentioned experiment as template, obtains Ct value X
1; By Ct value X
1bring reference gene typical curve formula 1 into, obtain the lg value of the copy number concentration value of the reference gene in transgene tobacco to be detected, i.e. Y
1;
The plasmid that contains foreign gene of gradient dilution of take is template, and upstream primer and the downstream primer of foreign gene of take is primer, carries out real-time fluorescence quantitative PCR amplification; The concentration of the plasmid template that contains foreign gene of extent of dilution minimum is denoted as to 1, calculate the ratio of the concentration of the concentration of other dilution plasmid templates that contain foreign gene and the plasmid template that contains foreign gene of extent of dilution minimum, by 1, be denoted as copy number concentration value with each ratio calculating; The lg value of copy number concentration value of take is ordinate zou, and corresponding Ct value, for X-coordinate drafting foreign gene typical curve, obtains foreign gene typical curve formula 2, and the while take the genomic dna of transgene tobacco to be detected and carries out above-mentioned experiment as template, obtains Ct value X
2; By Ct value X
2bring foreign gene typical curve formula 2 into, obtain the lg value of the copy number concentration value of the foreign gene in transgene tobacco to be detected, i.e. Y
2;
Y
2/ Y
1be the copy number of the foreign gene in transgene tobacco to be detected, work as Y
2/ Y
1after radix point, first is greater than at 5 o'clock, and integral part adds 1, is less than or equal at 5 o'clock, and integral part is constant, and integral part is the copy number of foreign gene in transgene tobacco;
The concentration unit of described template is identical;
Described reference gene is ribonucleotide reductase gene RNR2;
Described foreign gene is the gene proceeding in transgene tobacco.
4. method according to claim 3, is characterized in that: the concentration of the genomic dna of the non-transgenic tobacco of described gradient dilution is respectively 0.0406 μ g/ μ L, 0.00812 μ g/ μ L, 0.001624 μ g/ μ L, 0.0003248 μ g/ μ L, 0.00006496 μ g/ μ L.
5. according to the method described in claim 3 or 4, it is characterized in that: the reaction system of described real-time fluorescence quantitative PCR is: template 2 μ L, 10 μ molL
-1each 0.3 μ L of upstream primer, downstream primer,
premix Ex Taq
tMiI10 μ L, adds ddH
2o supplies system to 20 μ L;
premix Ex Taq
tMiI is specifically purchased from precious biotechnology (Dalian) company limited;
The reaction system of described real-time fluorescence quantitative PCR is: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 30s, 40 circulations.
6. according to the arbitrary described method of claim 3-5, it is characterized in that: described non-transgenic tobacco is non-transgenic tobacco bred K346.
7. primer claimed in claim 1, the test kit claimed in claim 2 application in copy number of foreign gene in detecting transgene tobacco.
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| CN105385687A (en) * | 2015-12-24 | 2016-03-09 | 中国烟草总公司郑州烟草研究院 | Cloning method for tobacco 26S RNA reference genes and application thereof |
| CN106191216A (en) * | 2015-04-30 | 2016-12-07 | 嘉和生物药业有限公司 | A kind of method of copy number of foreign gene in quantitative PCR detection transgenic cell |
| CN106755537A (en) * | 2017-03-02 | 2017-05-31 | 云南纳博生物科技有限公司 | A kind of method of the accurate detection chloroplast transformation degree of homogenization |
| CN106755444A (en) * | 2016-12-31 | 2017-05-31 | 东北农业大学 | A kind of soybean gene copy number analysis of variance method |
| CN107287282A (en) * | 2016-04-13 | 2017-10-24 | 上海杰隆生物工程股份有限公司 | A kind of method and its application of Rapid identification homozygous transgenic sheep |
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| CN106191216A (en) * | 2015-04-30 | 2016-12-07 | 嘉和生物药业有限公司 | A kind of method of copy number of foreign gene in quantitative PCR detection transgenic cell |
| CN105385687A (en) * | 2015-12-24 | 2016-03-09 | 中国烟草总公司郑州烟草研究院 | Cloning method for tobacco 26S RNA reference genes and application thereof |
| CN105385687B (en) * | 2015-12-24 | 2018-06-15 | 中国烟草总公司郑州烟草研究院 | The cloning process of tobacco 26S RNA reference genes and its application |
| CN107287282A (en) * | 2016-04-13 | 2017-10-24 | 上海杰隆生物工程股份有限公司 | A kind of method and its application of Rapid identification homozygous transgenic sheep |
| CN106755444A (en) * | 2016-12-31 | 2017-05-31 | 东北农业大学 | A kind of soybean gene copy number analysis of variance method |
| CN106755537A (en) * | 2017-03-02 | 2017-05-31 | 云南纳博生物科技有限公司 | A kind of method of the accurate detection chloroplast transformation degree of homogenization |
| CN106755537B (en) * | 2017-03-02 | 2021-04-02 | 云南纳博生物科技有限公司 | Method for accurately detecting chloroplast transformation homogenization degree |
| CN111206075A (en) * | 2020-02-26 | 2020-05-29 | 北京华安科创生物技术有限公司 | Method and primer for detecting copy number of recombinant human nerve growth factor gene |
| CN116397040A (en) * | 2022-10-27 | 2023-07-07 | 中国热带农业科学院三亚研究院 | Single copy papaya gene and method for detecting copy number of exogenous gene in transgenic papaya by using same |
| CN116397040B (en) * | 2022-10-27 | 2023-11-07 | 中国热带农业科学院三亚研究院 | Single copy papaya gene and method for detecting copy number of exogenous gene in transgenic papaya by using same |
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