CN105755111B - Method for quantitatively detecting content of MON757 transformant in cotton material population - Google Patents
Method for quantitatively detecting content of MON757 transformant in cotton material population Download PDFInfo
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- 229920000742 Cotton Polymers 0.000 title claims abstract description 97
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- 101150002976 ACP1 gene Proteins 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 238000003753 real-time PCR Methods 0.000 claims abstract description 17
- 239000012634 fragment Substances 0.000 claims abstract description 11
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- 239000000523 sample Substances 0.000 claims description 36
- 238000001514 detection method Methods 0.000 claims description 20
- 238000004925 denaturation Methods 0.000 claims description 8
- 230000036425 denaturation Effects 0.000 claims description 8
- 238000011088 calibration curve Methods 0.000 claims description 7
- 101150081640 CP1 gene Proteins 0.000 claims description 5
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- 241000196324 Embryophyta Species 0.000 description 12
- 235000012343 cottonseed oil Nutrition 0.000 description 9
- 108700019146 Transgenes Proteins 0.000 description 8
- 238000007859 qualitative PCR Methods 0.000 description 8
- 241000607479 Yersinia pestis Species 0.000 description 7
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Abstract
The invention discloses a method for quantitatively detecting the content of MON757 transformants in a cotton material group, which belongs to the technical field of biology and comprises the following steps: (1) obtaining a curve equation of a specific fragment sequence of the MON757 transformant, (2) obtaining a standard curve equation of an ACP1 gene of a standard gene in cotton, (3) randomly selecting a plurality of materials in a cotton material group to be detected, grinding, mixing and extracting DNA to be detected, (4) carrying out fluorescent quantitative PCR reaction on the MON757 transformant by taking the DNA to be detected as a template to measure the copy number of the MON757 transformant in the DNA to be detected; carrying out fluorescence quantitative PCR reaction on the ACP1 gene to obtain the copy number of the ACP1 gene in the DNA to be detected; (5) the content of the MON757 transformant is (copy number of the MON757 transformant/copy number of the ACP1 gene) multiplied by 2, the invention provides convenience for specifically identifying the existence and the content of the transgenic insect-resistant cotton MON757 transformant in the colony, and has the advantages of high efficiency, rapidness, high sensitivity, good specificity, low required experimental condition and very high practical value.
Description
Technical field
The invention belongs to field of biotechnology.Contain more particularly to MON757 transformant in quantitative detection cotton material group
The method of amount.
Background technique
Transgene cotton is one of primary transgenic crop of grown worldwide and the maximum transgenosis of China's cultivated area
Crop.2014,3,900,000 hectares of transgene cotton of 7,100,000 Cotton Varieties by Small Farming Households of China, the total cultivated area 93% of cotton is accounted for, it is higher than
2013 90% ratios.The transgene cotton of the commercial growth of China's approval at present mainly have it is domestic research and develop turn cry1Ab/
The transgenosis cry1Ac gene of the Insect Resistant Cotton and Monsanto Company of Ac fusion Insect Resistant Cotton MON531 transformant (http: //
www.moa.gov.cn/ztzl/zjyqwgz/spxx/)。
Transgene cotton MON757 transformant is researched and developed by Monsanto Company, is carried using the same plasmid of MON531 transformant
Body is converted.MON757 transformant and MON531 transformant the insertion point of cotton gene group, Insert Fragment structure not
Identical (https: //www.aphis.usda.gov/brs/aphisdocs/94_30801p.pdf).Currently, MON757 is converted
Body plants in approvals such as the U.S., Canada, Australia, New Zealand, Japan, South Korea and South Africa or allows to be used as food and feed former
(http://www.isaaa.org/gmapprovaldatabase/event/default.asp? EventID=55), but
China not yet gets the Green Light plantation as transformant.However, Wang Qiao etc. 2011 transgene cottons in detection China market
Shi Faxian, the miscellaneous cotton in transgenic cotton against pests Hubei Province No. 1 has phase with new cotton 33B (derived varieties that new cotton 33B is MON531 transformant)
With building, but transformation event is not identical, and the flanking sequence of the miscellaneous cotton in Hubei Province No. 1 is obtained using the method for Genome Walking,
And establish specificity of transformant qualitative checking method.
This laboratory found in accidental experiment by sequence alignment, Hubei Province No. 1 flanking sequence of miscellaneous cotton and patent US
The height of MON757 transformant sequence disclosed in 6893826 is similar, thus may determine that the transgenosis transformant of the miscellaneous cotton in Hubei Province No. 1 is come
Derived from MON757 transformant.
In the miscellaneous cotton in Hubei Province No. 1 detect MON757 transformant also illustrate with MON757 transformant be parent obtain breeding progeny
It is spread to China.It is the important finger for detecting transgene cotton quality to the assay of MON757 transformant in cotton material
Mark, or the mark of transgenosis provides technical basis.But at home and abroad there is no MON757 transformant quantitative PCR sides at present
The report of method.
Summary of the invention
For the blank in above-mentioned field, whether the present invention provides one kind and can detect in cotton to be measured containing MON757 conversion
The kit and method of body and its content, technical solution disclosed by the invention are as follows:
The method of MON757 transformant content, includes the following steps: in a kind of quantitative detection cotton material group
(1) calibration curve equation of MON757 specificity of transformant fragment sequence is obtained,
The curvilinear equation is after being diluted to gradient concentration using the genomic DNA of MON757 transformant homozygosis material as mould
Plate carries out MON757 transformant quantitative fluorescent PCR to the template of the gradient concentration using the logarithm of gradient concentration as abscissa
Reacting resulting fluorescence signal value is ordinate, fits curvilinear equation；
The primer and probe that the MON757 transformant quantitative fluorescent PCR reaction uses is as follows:
757-3QF:5 '-GGCATGCACATACAAATG-3 ',
757-3QR:5 '-CCCATTATTATTCTGTTGTTG-3 ',
Probe:
757-3QP:5 '-FAM-ACGAACGGATAAACCCTTTCTGGA-BHQ1-3 '；
(2) calibration curve equation of cotton internal standard Gene A CP1 gene is obtained:
The curvilinear equation is using the curvilinear equation institute for obtaining MON757 specificity of transformant fragment sequence in step (1)
Using DNA profiling, using the log concentration value of template as abscissa, ACP1 gene by fluorescence quantitative PCR reaction gained is carried out to template
Fluorescence signal value be ordinate, the equation for drawing up out；
Primer, probe, PCR reaction system and the program that wherein the quantitative fluorescent PCR reaction uses use national standard
The method of documented quantitative detection ACP1 gene in " No. 1943 bulletin -1-2013 of the Ministry of Agriculture "；
(3) multiple materials in cotton material group to be measured are randomly selected, DNA to be measured is extracted in mixing after grinding,
(4) reaction of MON757 transformant quantitative fluorescent PCR is carried out by template of DNA to be measured, reads fluorescence signal value and band
The copy of MON757 transformant in DNA to be measured is calculated in the curvilinear equation for entering the MON757 specificity of transformant fragment sequence
Number；
The ACP1 gene by fluorescence quantitative PCR reaction is carried out by template of DNA to be measured, fluorescence signal value is read and brings cotton into
The copy number of ACP1 gene in DNA to be measured is calculated in the calibration curve equation of flower internal standard Gene A CP1 gene；
(5) MON757 transformant content=(MON757 transformant copy number/ACP1 gene copy number) × 2.
The system of the preferably described MON757 transformant quantitative fluorescent PCR reaction are as follows: 10 μ L of Premix Ex Taq TM,
The 10 μm of upstream and downstream ol/L primer each 1.5 μ L, 10 μm of ol/L probes 0.75 μ L, 50 × ROX 0.4 μ L, 2 μ L of DNA profiling, are mended ultrapure
Water is to 20 μ L of total volume；
The program of MON757 transformant quantitative fluorescent PCR reaction are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of denaturation 5s, 60 DEG C are moved back
Fire and extension 34s, amplified reaction carries out 40 circulations, in 60 DEG C of collection PCR amplification fluorescence signals.
Since transgenic cotton against pests MON757 transformant has been approved by nearly 20 years of plantation, the cotton of import in China in U.S. etc.
It is difficult to have avoided mentioned component in colored and its converted products.And this is also detected that in the transgene cotton of China, provinces and regions, part
A transformant.
The present invention is had found by sequence alignment, disclosed in Hubei Province No. 1 flanking sequence of miscellaneous cotton and patent US 6893826
MON757 transformant sequence height is similar, thus may determine that the transgenosis transformant of the miscellaneous cotton in Hubei Province No. 1 is converted from MON757
Body.
The present invention also for target, devises quantitative PCR and draws with MON757 transformant connection region sequence (Seq ID No.9)
Object and probe, and the quantitative PCR detecting method of MON757 specificity of transformant is established, it is suitble to MON757 in cotton batch sample
The assay of transformant.Based on experimental data, the present invention provides turn for MON757 in quantitative detection cotton batch sample
Change the detection method and kit of the content of body.
To sum up, therefore the present invention is that quantitative determination of specificity transgenic cotton against pests MON757 transformant is provided convenience, this
The advantages of a little methods, is that quick, high sensitivity, specificity is good, required experiment condition is not high, therefore has very high practical valence
Value.
Detailed description of the invention
The primer and probe schematic diagram of 757 transformant quantitative PCR detection of Fig. 1 transgene cotton MON
Lowercase is insetion sequence in sequence, and capitalization is cotton gene group sequence
The qualitative PCR of Fig. 2 .MON757 transformant detects
1-6:MON757 homozygous sample；7-14:MON757 heterozygosis sample；15-20: non-transgenic sample；21-22: blank
Control；M:DL2000
The MON757 transformant qualitative PCR of Fig. 3 simple grain transgenic cotton floral material detects
A. F1 B. China favour 4 of the miscellaneous cotton in Hubei Province No. 7
Fig. 4 quantifying PCR method amplification curve and standard curve
A.MON757 transformant；B.ACP1 gene
Specific embodiment
Present invention will be further explained below with reference to specific examples.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as
The molecular cloning of Sambrook etc.: laboratory manual (New York:Cold Spring Harbor Laboratory Press,
2001) condition described in, or according to condition proposed by instrument or reagent manufacturer.
The design of 1. primer and probe of embodiment
The present invention accidentally passes through sequence alignment discovery, the miscellaneous cotton in Hubei Province No. 1 flanking sequence (its full length sequence Genbank accession number
It is similar to the height of MON757 transformant sequence disclosed in patent US 6893826 for AR656168, overall length 4973bp), therefore can
To determine the transgenosis transformant of the miscellaneous cotton in Hubei Province No. 1 from MON757 transformant.
The design of qualitative detection primer:
The present invention designs on design primer and insetion sequence on the genome of MON757 transformant insertion point two sides to draw
Object, three primers are as follows:
757-GF:5 '-CTAACCTCAATGAGAGCTACC-3 ' (Seq ID No.1),
757-TF:5 '-TCATGTAGCAATGTCCTGCC-3 ' (Seq ID No.2),
757-GR:5 '-GTAATAACTTGATGATGATTTGAG-3 ' (Seq ID No.3), as shown in Figure 1.
Wherein 757-GF/757-TF amplification region is 5 ' end cotton gene group and transgenic sequences of MON757 transformant
Bonding pad, extension increasing sequence of the 757-GF/757-TF to MON757 transformant, 184bp, as shown in Seq ID No.4；
757-GF/757-GR amplified fragments are cotton gene group sequence, are located at the Dt_ of cotton gene group (upland cotton)
The position 10276759-10277039 of chr11 chromosome, extension increasing sequence 291bp.And in the genome containing transformant, it inserts
Segment between the primer of angle of striking is the region for including entire insert, it is contemplated that more than 10kb, so long segment is general
It cannot then be expanded under the conditions of logical PCR；Therefore it is only capable of in the sample without MON757 transformant, obtains cotton gene group
The amplified production of the 291bp of sequence, as shown in Seq ID No.5.；
Three primers in combination use, it is contemplated that two band can be amplified in heterozygote.
It can be used for conventional PCR method and quick and precisely identify that the MON757 in field cotton single plant or simple grain cotton seeds is converted
The pure heterozygous state of body.
The design of quantitative detection primer and probe:
The present invention is with bonding pad (the Seq ID No.9 of the transgenic sequence of MON757 transformant and 3 ' end cotton gene groups
Shown, full length sequence Genbank accession number is AR656167, overall length 513bp) it is target, it devises and is converted for MON757
The primer and probe of the quantitative PCR detecting method of body specificity is suitble to measure containing for MON757 transformant in cotton batch sample
It is fixed.It is specific as follows:
2 primers are as follows: 757-3QF:5 '-GGCATGCACATACAAATG-3 ' (Seq ID No.6), 757-3QR:5 '-
CCCATTATTATTCTGTTGTTG-3 ' (Seq ID No.7),
Probe are as follows: 757-3QP:5 '-FAM-ACGAACGGATAAACCCTTTCTGGA-BHQ1-3 ' (Seq ID No.8).
The qualitative PCR detection method of 2. transgenic cotton against pests MON757 transformant of embodiment
1 experimental material
1.1 transgenic pest-resistant cotton materials
Hubei Province miscellaneous cotton No. 1 (Run Fumian 839) (raw manufacturer: scented rice industry Co., Ltd in Hubei, product batch number: 2009-
10, authorization number: cotton 001-2000 is examined in Hubei Province), it is purchased from Hubei Province's cotton seeds market within 2010.In Chinese Academy of Agricultural Sciences plant
Protective strategy institute chamber planting obtains the homozygous material and heterozygosis of MON757 transformant through single plant screening and identification, self propagated etc.
Material.
Hubei Province No. 7 F1 of miscellaneous cotton (the macro miscellaneous cotton 071 of alias) (raw manufacturer: Cang Gu Man Zhongye Co., Ltd, Hubei Province, authorization number:
Cotton 2004003 is examined in Hubei Province), it is purchased from Hubei Province's cotton seeds market within 2014.
Magnificent favour 4 (raw manufacturer: Hubei Huimin Agriculture Technology Co, ltd, product batch number: 201310, authorization number: state examines
Cotton 2000010), it is purchased from Hubei Province's cotton seeds market within 2014.
1.2 reagent
Molecular biology reagents, such as dNTPs, Taq archaeal dna polymerase, DL2000Marker are purchased from Dalian treasured bioengineering
Co., Ltd.
Other biochemical reagents are that import packing or domestic analysis are pure.Primer wins biotechnology Limited Liability public affairs by Beijing three
Department's synthesis.
1.3 laboratory apparatus
PCR amplification instrument: model S1000 (Bio-Rad company)
Nucleic acid electrophoresis apparatus: DYY-III type nucleic acid electrophoresis apparatus (Liuyi Instruments Plant, Beijing)
Gel imaging system: BiospectrumAC (UVP company)
Other Instruments includes: centrifuge, constant temperature heating plate, electronic balance, incubator etc..
2 experimental methods and process
2.1 sample pre-treatments
The cotton material of chamber planting, takes single-strain blade respectively.Hubei Province miscellaneous cotton No. 4 samples of No. 7 F1 and Hua Hui randomly select 60
Grain seed, simple grain grinding.
2.2 cotton genomic dnas extract
According to the operation manual of plant DNA extraction kit (TIANGEN Bioisystech Co., Ltd), cotton material is carried out
The extraction of total DNA.The DNA solution for taking 5 μ l to extract, with 0.8% agarose gel electrophoresis, according to its brightness and banding pattern come preliminary
The quality of DNA is extracted in judgement.The concentration and purity of extracted DNA are measured using ultraviolet specrophotometer.
The detection of 2.3 chamber planting single plant cotton materials
Using the MON757 transformant qualitative PCR detection of foundation being made of 3 primers 757-GF, 757-GR and 757-TR
Method, selection homozygosis, heterozygosis and the greenhouse cotton material single plant without MON757 transformant are verified.
PCR reaction system: GoGreenMaster Mix 10 μ L, primer 757-GF, 757-GR and 757-TR (10 μ
Mol/L) each 1 μ L, 2 μ L of DNA profiling mend ultrapure water to 20 μ L of total volume.Response procedures: 95 DEG C of initial denaturation 4min, 95 DEG C of denaturation
30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, amplified reaction carry out 35 circulations, 72 DEG C of heat preservation 10min.
In MON757 transformant heterozygosis sample, three primers expand the cotton gene group sequence and 5 ' of insertion point respectively
Region sequence is connected, the two kinds of amplified productions of 291 and 184bp are obtained；In MON757 transformant homozygous sample, it is only capable of acquisition 5 ' even
Connect the amplified production of the 184bp of region sequence, and the segment between Genome Primer be include entire insert region (it is expected that
More than 10kb), it cannot then be expanded under the conditions of regular-PCR；And in the sample without MON757 transformant, it is only capable of obtaining cotton
The amplified production of the 291bp of flower genome sequence.
Verification result shows as shown in Figure 1, homozygosis MON757 transformant plant only expands and obtains the segment of a 184bp,
Heterozygous plant obtains two band of 184bp and 291bp, and the plant without MON757 transformant has to the band of a 291bp,
This is completely the same with expected results.Therefore, the MON757 transformant qualitative PCR method of foundation can effectively identify MON757 conversion
Body and its existence.
The qualitative PCR detection of MON757 transformant in 2.4 cotton seeds samples
60 simple grains for randomly selecting Hubei Province No. 7 F1 of miscellaneous cotton and No. 4 samples of Hua Hui respectively, using the MON757 transformant of foundation
Specific qualitative PCR method is identified (Fig. 3), the results showed that MON757 transformant homozygosis, heterozygosis in No. 7 F1 samples of miscellaneous cotton of Hubei Province
It is respectively 0,2 and 58 with what is do not contained, and these three types of in No. 4 samples of magnificent favour is respectively 6,22 and 32.Referring to national standard
The calculation method of transgenic content is calculated:
MON757 transformant content=(homozygous MON757 transformant quantity+heterozygosis MON757 transformant quantity/2)/60.
Calculated result shows that the MON757 transformant content of Hubei Province miscellaneous cotton No. 4 samples of No. 7 F1 and Hua Hui is respectively 1.67% He
28.33%.
The quantitative PCR detecting method of 3. transgenic cotton against pests MON757 transformant of embodiment
1 experimental material
1.1 transgenic pest-resistant cotton materials
Hubei Province miscellaneous cotton No. 1 (Run Fumian 839) (raw manufacturer: scented rice industry Co., Ltd in Hubei, product batch number: 2009-
10, authorization number: cotton 001-2000 is examined in Hubei Province), it is purchased from Hubei Province's cotton seeds market within 2010.
The homozygous material of MON757 transformant: the miscellaneous cotton in Hubei Province No. 1 is in Plant Protection institute, Chinese Academy of Agricultral Sciences's greenhouse kind
It plants, the homozygous material and hybrid material of MON757 transformant is obtained through single plant screening and identification, self propagated etc., be used for quantitative detection
The preparation of method standard curve.
Hubei Province No. 7 F1 of miscellaneous cotton (the macro miscellaneous cotton 071 of alias) (raw manufacturer: Cang Gu Man Zhongye Co., Ltd, Hubei Province, authorization number:
Cotton 2004003 is examined in Hubei Province), it is purchased from Hubei Province's cotton seeds market within 2014.
Magnificent favour 4 (raw manufacturer: Hubei Huimin Agriculture Technology Co, ltd, product batch number: 201310, authorization number: state examines
Cotton 2000010), it is purchased from Hubei Province's cotton seeds market within 2014.
1.2 reagent
Molecular biology reagents, such as Premix Ex Taq TM (Probe qPCR) are limited purchased from Dalian treasured bioengineering
Company.
Other biochemical reagents are that import packing or domestic analysis are pure.It is limited that primer and probe by Beijing three wins biotechnology
Responsible company's synthesis.
1.3 laboratory apparatus
Quantitative pcr amplification instrument: model ABI7500 (ABI company)
Other Instruments includes: centrifuge, constant temperature heating plate, electronic balance, incubator etc..
2 experimental methods and process
2.1 sample pre-treatments
Hubei Province miscellaneous cotton No. 4 samples of No. 7 F1 and Hua Hui randomly select 600 seeds, are ground.
2.2 cotton genomic dnas extract
According to the operation manual of plant DNA extraction kit (TIANGEN Bioisystech Co., Ltd), cotton material is carried out
The extraction of total DNA.The DNA solution for taking 5 μ l to extract, with 0.8% agarose gel electrophoresis, according to its brightness and banding pattern come preliminary
The quality of DNA is extracted in judgement.The concentration and purity of extracted DNA are measured using ultraviolet specrophotometer.
The foundation of 3 quantifying PCR method standard curves
The Genomic DNA solution of MON757 transformant homozygosis material is successively diluted to ultrapure water every microlitre 22000,
2200,220,44,22 cotton gene group copies.Using the logarithm of template copy numbers as abscissa, built using Cq value as ordinate
The standard curve of vertical MON757 specificity of transformant fragment sequence and cotton internal standard gene.
The quantitative PCR reaction system of MON757 transformant: Premix Ex Taq TM (Probe qPCR) 10 μ L, upstream and downstream
Primer (10 μm of ol/L) each 1.5 μ L, probe (10 μm of ol/L) 0.75 μ L, 50 × ROX 0.4 μ L, 2 μ L of DNA profiling, mends ultrapure water
To 20 μ L of total volume.
It is expanded using 7500 fluorescence quantitative PCR instrument of ABI PRISM (U.S.), response procedures: 95 DEG C of initial denaturation 2min, 95
DEG C denaturation 5s, 60 DEG C annealing and extend 34s, amplified reaction carry out 40 circulation.PCR amplification fluorescence signal is collected at 60 DEG C.
The quantitative pcr amplification standard curve of the MON757 specificity of transformant of foundation.The equation of linear regression of standard curve
For y=-3.456x+39.000, the linear coefficient of determination (R2) is 0.998, and amplification efficiency 94.7%, wherein y is Cq value, and x is
The logarithm (Fig. 4, A) of copy number.
Referring to national standard " No. 1943 bulletin -1-2013 of the Ministry of Agriculture ", with every microlitre 11000,1100,110,22,11
The MON757 transformant homozygosis material genomic DNA of cotton gene group copy is template, with national standard " No. 1943 public affairs of the Ministry of Agriculture
Announcement -1-2013 " disclosed in ACP1 gene primer, probe, PCR program and system prepare calibration curve equation, and y is Cq value,
X is the logarithm of copy number, and the standard curve regression equation of the cotton internal standard Gene A CP1 gene of foundation is y=-3.553x+
40.494, the linear coefficient of determination (R2) is 0.999, and amplification efficiency is 91.2% (Fig. 4, B).
MON757 transformant and the standard curve parameter of ACP1 gene comply fully with real time fluorescent PCR method Good Laboratory
It is required that can be used for quantitative detection.
The quantitative PCR detection of MON757 transformant in 2.4 cotton seeds samples
MON757 in miscellaneous cotton No. 4 samples of No. 7 F1 and Hua Hui of Hubei Province is measured using the MON757 transformant quantifying PCR method of foundation
The copy number of transformant and ACP1 gene.
Using primer/probe the (the 6th of ACP1 gene disclosed in national standard " No. 1943 bulletin -1-2013 of the Ministry of Agriculture "
Page A2.1), the copy number of PCR program and system (the 7.5.2 section of page 4) measurement ACP1 gene.
Since the copy number of the ACP1 gene in cotton gene group is 2, so cotton gene group copy number is in sample
The 1/2 of the copy number of ACP1 gene.Therefore the content of MON757 transformant in sample is quantitatively calculated according to following formula.
MON757 transformant content=(MON757 transformant copy number/ACP1 gene copy number) * 2
Testing result shows Hubei Province No. 7 F1 of miscellaneous cotton and Hua Hui 4 MON757 transformant contents are respectively 1.56% He
25.88%, this coincide compared with identifying the 1.67% and 28.33% of the pure heterozygosis estimation of simple grain by qualitative PCR, illustrates quantitatively to examine
The testing result of survey method is accurate and reliable.
Claims (6)
1. the method for MON757 transformant content, includes the following steps: in a kind of quantitative detection cotton material group
(1) curvilinear equation of MON757 specificity of transformant fragment sequence is obtained,
The curvilinear equation is after being diluted to gradient concentration using the genomic DNA of MON757 transformant homozygosis material as template,
Using the logarithm of gradient concentration as abscissa, it is anti-that MON757 transformant quantitative fluorescent PCR is carried out to the template of the gradient concentration
Answering resulting fluorescence signal value is ordinate, fits the equation come；
The primer and probe that the MON757 transformant quantitative fluorescent PCR reaction uses is as follows:
757-3QF:5 '-GGCATGCACATACAAATG-3 ',
757-3QR:5 '-CCCATTATTATTCTGTTGTTG-3 ',
Probe:
757-3QP:5 '-FAM-ACGAACGGATAAACCCTTTCTGGA-BHQ1-3 '；
(2) calibration curve equation of cotton internal standard Gene A CP1 gene is obtained:
The curvilinear equation is to be diluted to ladder using the genomic DNA of identical MON757 transformant homozygosis material in step (1)
It spends after concentration and ACP1 gene by fluorescence quantitative PCR is carried out to template and reacts institute using the log concentration value of template as abscissa for template
The fluorescence signal value obtained is ordinate, fits the equation come；
Primer, probe, PCR reaction system and the program that wherein the quantitative fluorescent PCR reaction uses are using national standard " agricultural
Bulletin-the 1-2013 of portion 1943 " in documented quantitative detection ACP1 gene method；
(3) multiple materials in cotton material group to be measured are randomly selected, DNA to be measured is extracted in mixing after grinding,
(4) reaction of MON757 transformant quantitative fluorescent PCR is carried out by template of DNA to be measured, read fluorescence signal value and brings institute into
The copy number of MON757 transformant in DNA to be measured is calculated in the curvilinear equation for stating MON757 specificity of transformant fragment sequence；
The ACP1 gene by fluorescence quantitative PCR reaction is carried out by template of DNA to be measured, fluorescence signal value is read and brings into cotton
The copy number of ACP1 gene in DNA to be measured is calculated in the calibration curve equation of standard gene ACP1 gene；
(5) MON757 transformant content=(MON757 transformant copy number/ACP1 gene copy number) × 2.
2. according to the method described in claim 1, the system of MON757 transformant quantitative fluorescent PCR reaction are as follows: Premix
Ex Taq TM 10 μ L, the 10 μm of upstream and downstream ol/L primer each 1.5 μ L, 10 μm of ol/L probes, 0.75 0.4 μ L of μ L, 50 × ROX,
2 μ L of DNA profiling mends ultrapure water to 20 μ L of total volume；
The program of MON757 transformant quantitative fluorescent PCR reaction are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of denaturation 5s, 60 DEG C of annealing with
Extend 34s, amplified reaction carries out 40 circulations, in 60 DEG C of collection PCR amplification fluorescence signals.
3. according to the method described in claim 2, the curvilinear equation of the MON757 specificity of transformant fragment sequence are as follows:
Y=-3.456x+39.000, R2=0.998.
4. a kind of kit for MON757 transformant content in fluorescence quantifying PCR method quantitative detection cotton material group,
It is characterised in that it includes the primer and probe of quantitative fluorescent PCR reaction, sequence are as follows:
757-3QF:5 '-GGCATGCACATACAAATG-3 ',
757-3QR:5 '-CCCATTATTATTCTGTTGTTG-3 ',
Probe are as follows:
757-3QP:5 '-FAM-ACGAACGGATAAACCCTTTCTGGA-BHQ1-3 '.
5. kit according to claim 4 further includes (1) curvilinear equation specification,
Record the curvilinear equation y=-3.456x+39.000, R2=0.998 of MON757 specificity of transformant fragment sequence；
And the calibration curve equation y=-3.553x+40.494, R2=0.999 of cotton internal standard Gene A CP1 gene.
6. kit according to claim 4 or 5 further includes the MON757 as standard items for being used to prepare standard curve
The genomic DNA of transformant homozygosis material.
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| 转基因棉花MON757转化体特异性PCR检测方法及应用;杨阳等;《农业生物技术学报》;20161231;第24卷(第6期);908-918 * |
| 转基因植物及其产品成分检测 棉花内标准基因定性PCR方法;中华人民共和国农业部公报;《中华人民共和国农业部公告第1943号》;20130523;全文 * |
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