CN101792814A - PCR method for detecting specificity of transformant of transgenic pest-resistant cotton GK12 - Google Patents
PCR method for detecting specificity of transformant of transgenic pest-resistant cotton GK12 Download PDFInfo
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Abstract
The invention relates to a PCR method for detecting the specificity of the transformant of transgenic pest-resistant cotton GK12, which belongs to the field of biotechnology. The method has the following characteristics that: the forward primer in PCR reaction is designed according to a 1-546 position sequence of SEQ ID NO1, and the reverse primer is designed according to a 547-1625 position sequence of SEQ ID NO1. The method can be an effective means for identifying transgenic pest-resistant cotton strain GK12 and transgenic cotton strain with the variety as parent. The method has the advantages of quickness, high sensitivity, good specificity and low needed experiment conditions, and therefore is highly practical.
Description
Technical field
The invention belongs to biological technical field.Particularly relate to a kind of specificity of transformant PCR method of transgenic pest-resistant cotton GK 12 and specificity of transformant sequence of dependence thereof differentiated.
Background technology
China is one of main plantation country of genetically modified crops, and cultivated area was positioned at the 6th in the whole world in 2009.The genetically modified crops that China has ratified commercialization plantation comprise the transgenic papaya of transgenic pest-resistant cotton, anti-pawpaw ring spot virus etc.Wherein, soil of transgenic Bt cotton is the genetically modified crops of China's cultivated area maximum, and cultivated area reached 3,700,000 hectares in 2009, accounts for nearly 70% of Cotton in China total cultivated area.In the transgenic cotton against pests of China's plantation at present extensively, be that two big important masters plant strain with the state of the MON531 transformant of Monsanto company (as new cotton 33B etc.) and Biological Technology institute, Chinese Academy of Agricultural Sciences's development anti-serial (as GK12 etc.).Specificity of transformant sequence and the specificity of transformant detection method of MON531 have had report at present, but specificity of transformant sequence and the specificity of transformant detection method of one of representative strain of the anti-series of state GK12 are not appeared in the newspapers as yet.
At present, round pcr is the technology that is most widely used during genetically modified crops detect.When using this technology and carry out the genetically modified crops qualitative detection, it is divided into four classes according to the target zone and the specificity of its amplification: one, screening detection, exogenous promoter and the terminator general with transgenosis are the target zone; Two, gene specific detects, and is the target zone with special external source goal gene; Three, make up specific detection, with the special target zone that is configured between the transgenosis element; Four, specificity of transformant detects, and is the target zone with specificity of transformant fragment (external source is inserted fragment and acceptor gene group connecting zone).This four classes detection method increases gradually to the detection specificity of genetically modified crops, it is at present to the highest detection method of transgenic strain detection specificity that specificity of transformant detects, also be the most frequently used identity detection method during present transgenosis detects, this detection method can be distinguished the different transformed plant strains that same conversion carrier produces.
Summary of the invention
At the blank in the above-mentioned field, the invention provides the specificity of transformant sequence of transgenic pest-resistant cotton GK 12 and the specificity of transformant PCR method of differentiating GK12, this PCR method can utilize common PCR instrument, reagent quick and precisely to identify transgenic pest-resistant cotton GK 12, and is parent's cotton strain with GK12.
Transgenic pest-resistant cotton GK 12 specificity of transformant sequence, shown in Seq ID No.1, wherein the conversion carrier sequence is positioned at 1-546bp, and cotton gene group sequence is positioned at 547-1625bp.
The PCR method for detecting specificity of transformant of transgenic cotton GK12 is characterized in that: the forward primer in the PCR reaction is according to 1-546 bit sequence design among the SEQ ID NO1, and reverse primer is according to the 547-1625 bit sequence design of SEQ ID NO1.
PCR detection method according to claim 1,
Described forward primer is GK12-F:5 '-CCGCAATGTGTTGTTAAGT-3 ',
Described reverse primer is GK12-R:5 '-TTCCCACATGGCCAAAAG-3 '.
The specificity of transformant sequence of transgenic pest-resistant cotton GK 12 provided by the invention and the specificity of transformant PCR method that relies on its foundation can be used as a kind of evaluation transgenic cotton against pests strain GK12, and be the effective means of parent's transgenic cotton strain with this kind, because one of transgenic cotton against pests strain that transgenic pest-resistant cotton GK 12 is China extensively plants at present, therefore the present invention provides convenience for specificity evaluation transgenic pest-resistant cotton GK 12, the advantage of this method is fast, highly sensitive, specificity is good, required experiment condition is not high, and therefore very high practical value is arranged.
Description of drawings
The Genome Walking amplification of Fig. 1 transgenic pest-resistant cotton GK 12 specificity of transformant sequence
A: Walking result for the first time; B: Walking result for the second time
M:DNA Marker DL2000; The Walking product of 1-8:DL1-DL8
Fig. 2 transgenic cotton GK12 specificity of transformant PCR detected result
M:DNA Marker DL2000; 1:GK12,2-11:10 market cotton sample
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the molecular cloning of Sambrook etc. for example: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or the condition of being advised according to instrument or reagent manufacturer.
The clone of transgenic pest-resistant cotton GK 12 specificity of transformant sequence
1 experiment material
1.1 vegetable material
Transgenic cotton against pests: GK12, there is preservation in this laboratory, can provide to the public.
1.2 enzyme and reagent
Molecular biology reagent, as dNTPs, Taq archaeal dna polymerase, DL2000Marker etc. available from the precious biotechnology in Dalian company limited.
Other biochemical reagents are import packing or homemade analytical pure.Primer is synthetic by Beijing AudioCodes biotechnology limited liability company.
1.3 laboratory apparatus
Pcr amplification instrument: Mastercycle gradient (Eppendorf co.)
Nucleic acid electrophoresis apparatus: DYY-III type nucleic acid electrophoresis apparatus (Liuyi Instruments Plant, Beijing)
Dna sequencing instrument (the full-automatic fluorescence sequenator of Applied Biosystem377 type)
DNA electrophoretic analysis system: Kodak EDAS 290 (Kodak co.)
Other Instruments comprises: whizzer, thermostatically heating plate, electronic balance, incubator etc.
2 experimental techniques and process
2.1 cotton genomic dna extracts and detects
2.1.1 cotton genomic dna extracts
Get the material of the young tender cotton leaf of cotyledon period as DNA extraction, the operational manual according to pillar DNA of plants out test kit (day damp genome company) carries out the extraction of the total DNA of cotton material.
2.1.2DNA detect
Get the dna solution that 5 μ l extract, the agarose gel electrophoresis with 0.8% is tentatively judged the quality of extracting DNA according to its brightness and banding pattern.Adopt ultraviolet spectrophotometer to measure concentration and the purity of the DNA that is extracted.
2.2Genome walking separates the specificity of transformant sequence of transgenic pest-resistant cotton GK 12
Genome walking also is a kind of method of the known array side zone of ignorance sequence that increases, and key step is for the genomic dna enzyme is cut, is connected with joint, twice nest-type PRC amplification etc.Present embodiment adopts the method for Genome walking to obtain the specificity of transformant sequence of transgenic pest-resistant cotton GK 12.Concrete steps are with reference to Genome Walker
TMThe explanation of Universal Kit (Takara Dalian company) test kit, present embodiment have been carried out partly improving, and main operational steps is as follows.
2.2.1 the genomic dna enzyme is cut
Cut the GK12 genomic dna with Dra I, Stu I, Pvu II, EcoR V, Nru I, Pmac I, Sca I and Sma I enzyme respectively, be labeled as DL1～DL8.The enzyme system of cutting is: genomic dna (0.1 μ g/ μ L), 25 μ L; 10 * enzyme is cut Buffer, 10 μ L; Restriction endonuclease (10U/ μ L), 8 μ L; DdH
2O, 57 μ L; Cumulative volume 100 μ L.37 ℃ of temperature are bathed 2h, take out centrifuge tube, and low-speed centrifugal 5～10s continues temperature and bathes 16～18h.Respectively get 5 μ L and carry out agarose gel electrophoresis, detect enzyme and cut whether fully.
2.2.2 purifying enzyme is cut product
Cut product by reclaiming test kit specification sheets purifying enzyme.
2.2.3 ligation
The ligation system: the enzyme of purifying is cut product, 4 μ L; Joint (25 μ M), 1.9 μ L; 10 * connection Buffer, 1.6 μ L; T4DNA ligase enzyme (6U/ μ L), 0.5 μ L; DdH
2O, 8 μ L; Cumulative volume 16 μ L.
Mixed reaction solution, 16 ℃ of temperature are bathed and are spent the night, then 70 ℃ of sex change 5min.Every pipe adds 72 μ L ddH
2O, mixing ,-20 ℃ of preservations are standby.
2.2.4PCR reaction
Genome Walking needs two-wheeled PCR reaction altogether, and the reaction system of its PCR sees Table 1.
Table 1 reaction system (μ L)
| The first round | Second takes turns | |
| ??ddH2O | ??17.375 | ??36.25 |
| ??10×Buffer(Mg 2+Plus) | ??2.5 | ??5 |
| ??dNTP(2.5mM?each) | ??2 | ??4 |
| ??AP1(10μM) | ??1 | ??2 |
| ??GSP?Primer?1(10μM) | ??1 | ??2 |
| ??Ex?Taq(5U/μL) | ??0.125 | ??0.25 |
| ??Template(20ng/μL) | ??1 | (0.5 first round reaction product) |
| ??Total?volume | ??25 | ??50 |
The program of two-wheeled pcr amplification sees Table 2.
Table 2Genome Walking amplification program
2.2.5 sequencing and analysis
Pcr amplification product carries out electrophoresis on 0.8% agarose gel, adopt QIAquick Gel Extraction kit to reclaim amplified fragments, be connected to pGEM-T easy (Promega, Madison, Wis.), adopt ABI PRISM 1300Genetic Analyzer to carry out sequencing.Employing software vectorNTI10.0 (Invitrogen) splices the sequence of measuring and analyzes.In ncbi database (http://www.ncbi.nlm.nih.gov/), retrieve similar genome sequence with BLASTN.
3 experimental results
3.1 transgenic pest-resistant cotton GK 12 specificity of transformant sequencing
Specificity of transformant sequence through twice Genome Walking acquisition transgenic pest-resistant cotton GK 12.For the first time GenomeWalking carries out the first round and second respectively with primer GW1-P1 and GW1-P2 (table 3) and takes turns pcr amplification, and the result obtains the amplified production (Figure 1A) of 639bp.For the second time Genome Walking with the first time product sequence be basic design primer GW2-P1 and GW2-P2 (table 3), carry out the first round and second respectively and take turns pcr amplification, the result obtains the amplified production (Fig. 2 A) of 1115bp.
Table 3Genome Walking primer
| Primer | Sequence |
| ??GW1-P1 | ??5’-CGAAAGTACATTCGCCGTGGCTATGG-3’ |
| ??GW1-P2 | ??5’-GGAATACCCGGCAATGCAGCAG-3’ |
| ??GW2-P1 | ??5’-CGTCCGCAATGTGTTGTTAAGT-3’ |
| ??GW2-P2 | ??5’-GTCTAAGCGTCAATTTGTTTACATCAC-3’ |
It is the sequence of 1625bp that twice Genome Walking product sequence assembly obtains length.
3.2 transgenic pest-resistant cotton GK 12 specificity of transformant sequential analysis
Submit the 1625bp sequence that obtains to the ncbi database compare of analysis, wherein 1-546bp is the conversion carrier sequence, and 547-1625bp is a cotton gene group sequence.
Specificity of transformant PCR method of the present invention is applied to differentiate transgenic pest-resistant cotton GK 12
1 experiment material
1.1 vegetable material
Transgenic cotton against pests: GK12
10 cotton materials are available from market.
1.2 enzyme and reagent
Molecular biology reagent, as dNTPs, Taq archaeal dna polymerase, Marker etc. available from the precious biotinylated biomolecule in Dalian Engineering Co., Ltd.Other biochemical reagents are import packing or homemade analytical pure.Primer is synthetic by Beijing AudioCodes biotechnology limited liability company.
1.3 laboratory apparatus
Pcr amplification instrument: Mastercycle gradient (Eppendorf co.)
Nucleic acid electrophoresis apparatus: DYY-III type nucleic acid electrophoresis apparatus (Liuyi Instruments Plant, Beijing)
DNA electrophoretic analysis system: Kodak EDAS 290 (Kodak co.)
Other Instruments comprises: whizzer, thermostatically heating plate, electronic balance, incubator etc.
2 experimental techniques and process
2.1 cotton genomic dna extracts and detects
See that 2.1 cotton genomic dnas extract and detect among the embodiment 1.
2.2 differentiate the specificity of transformant PCR method of transgenic pest-resistant cotton GK 12
PCR method of the present invention is applied to differentiate whether contain transgenic cotton against pests strain GK12 in the cotton sample.Adopt primer GK12-F/GK12-R combination (sequence sees Table 4), detect 10 cotton samples, see Fig. 2 available from market.
Experimental result shows that transgenic pest-resistant cotton GK 12 energy specific amplification goes out the product of 150bp, in 10 market samples of detection 1 fragment that also can amplify identical size is arranged.
Clone GK12 and the amplified fragments that can amplify the market sample of 150bp respectively, carry out sequential analysis by " 2.2.5 sequencing and analysis " among the embodiment 1.The sequencing results shows that the amplified fragments sequence of market sample and the extension increasing sequence of GK12 are in full accord, and is also all in full accord with the 491-640 position of sequence SEQ ID NO1.
Detected result shows PCR method provided by the invention can identify whether contain transgenic pest-resistant cotton GK 12 in the cotton sample well.
The PCR reaction system is: 0.25 μ L rTaq (5U/ μ L), 5 μ L, 10 * PCR Buffer (Mg
2+Plus), 4 μ L dNTP (each 2.5mM), each 2 μ L (10 μ M) of primer, each 2 μ L (20ng/ μ L) of template add sterile purified water and supply volume to 50 μ L.
Amplification program is: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 35 circulations; 72 ℃ of 10min.
Table 4 is differentiated the specificity of transformant PCR primer of transgenic pest-resistant cotton GK 12
| Primer | Sequence |
| ??GK12-F | ??5’-CCGCAATGTGTTGTTAAGT-3’ |
| ??GK12-R | ??5’-TTCCCACATGGCCAAAAG-3’ |
SEQUENCE?LISTING
＜110〉Plant Protection institute, Chinese Academy of Agricultral Sciences
＜120〉PCR method for detecting specificity of transformant of transgenic pest-resistant cotton GK 12
<130>P10191/ZWB
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＜213〉the specificity of transformant sequence of transgenic pest-resistant cotton GK 12
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ggaatacccg?gcaatgcagc?agcgattgcc?gactgggtta?tatcagaccg?atctgaggat????60
gagatgtcat?tctgggaatg?gcgtaacaaa?ggcgttcccc?gcagggttga?tcaccactgc????120
ccccatctcg?acgttataga?gcaagatgga?aaatctgtac?cgtgatatta?ttatagaatc????180
ctgtatatca?gacataggaa?gcaatttatg?atatctatct?attctctgta?atattgaatt????240
acttatataa?gtttcatatt?ttataatagt?atatatatct?tggtatataa?atgtatttat????300
aactttaaat?aactcgtaaa?ataattataa?ctgtaatgac?tccgcgcaat?atttacacat????360
agacacacac?atcatctcat?tgatgcttgg?taataattgt?cattagattg?tttttatgca????420
tagatgcact?cgaaatcagc?caattttaga?caagtatcaa?acggatgtga?attcagtaca????480
ttaaaaacgt?ccgcaatgtg?ttgttaagtt?gtctaagcgt?caatttgttt?acatcacaat????540
atatccatct?cattgacttg?tatcttaaac?tatatttata?accctatggc?cattgcccta????600
tttcttaaac?tatatttata?accttttggc?catgtgggaa?gaaaatcaga?tcccattctt????660
ccacccatga?aaaacattca?aatatagtgt?aatgattatt?taatttatct?tttaattata????720
aggttagagt?ataacttcag?ttcataaaaa?tgaaatatat?tttataatta?attatttatc????780
cttttaaaaa?atacttttac?tcataaaaat?aaaatacact?tcccgtgata?ttagataata????840
tttctcaaac?tcgcataatg?atttcatgga?caattaaaca?ggttggataa?taatggatta????900
gatggtgttt?caacagtttc?aacttttctc?gaatagttca?ctttcctcaa?aattgggttg????960
tcttttgacc?accataaaca?aaatacatat?acacaaatta?atataaaagt?tgaaaaaagc????1020
aaagtggact?ataaagtaat?gggatactta?ggcatgtcac?acacgtcaag?gtcttgaaat????1080
aaacatttca?acaaacatcc?aaaaaattca?atggccgaat?aaaaggggag?cttccattac????1140
atacacatat?aatgaaccga?catcaagtca?cgtcaaactg?taacttgaaa?gtgcaccaat????1200
taggtccatt?tatgattgat?ttagttatgt?cctttggatt?ccctatgcat?tcaatcacat??1260
ttagcctaat?ttttaatatt?agttacttaa?aaacataact?tttgacgcct?ttcataaaag??1320
ggaacagata?aatggagtac?agtacctctg?ccgacacttc?gtatggagga?atagaaagca??1380
ataaaatgac?aaactgtcgc?tttcgtttcc?ccttttcaac?tttgcctctt?tacaaatgca??1440
aatacattct?aacttacaat?aactgctgtc?atgactgctg?aattttcagg?gtcatatgag??1500
attatttttt?tattcaacta?tgaatttttt?ttattggtca?ttccagtaat?cgagttttct??1560
cttccttgtt?cattaattgt?agatactttt?ttgatggttg?gattcgaacg?tcgacacgcc??1620
gttgc??????????????????????????????????????????????????????????????1625
<210>2
<211>19
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＜213〉forward primer GK12-F
<400>2
ccgcaatgtg?ttgttaagt???????????????????????????????????????????????19
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＜213〉reverse primer GK12-R
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ttcccacatg?gccaaaag????????????????????????????????????????????????18
<210>4
<211>26
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＜213〉Genome Walking primer GW1-P1
<400>4
cgaaagtaca?ttcgccgtgg?ctatgg???????????????????????????????????????26
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＜213〉Genome Walking primer GW1-P2
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ggaatacccg?gcaatgcagc?ag????????????????????????????????????????????22
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＜213〉Genome Walking primer GW2P1
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cgtccgcaat?gtgttgttaa?gt????????????????????????????????????????????22
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＜213〉Genome Walking primer GW2-P2
<400>7
gtctaagcgt?caatttgttt?acatcac???????????????????????????????????????27
Claims (2)
1. the PCR method for detecting specificity of transformant of transgenic pest-resistant cotton GK 12 is characterized in that: the forward primer in the PCR reaction is according to 1-546 bit sequence design among the SEQ ID NO1, and reverse primer is according to the 547-1625 bit sequence design of SEQ ID NO1.
2. PCR detection method according to claim 1,
Described forward primer is GK12-F:5 ' CCGCAATGTGTTGTTAAGT-3 ',
Described reverse primer is GK12-R:5 ' TTCCCACATGGCCAAAAG-3 '.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935705A (en) * | 2010-08-30 | 2011-01-05 | 中国农业科学院植物保护研究所 | PCR detection method of transformant specificity of transgenic insect-resistant cotton Hubei mixed cotton No.1 and application thereof |
| WO2012068772A1 (en) * | 2010-11-22 | 2012-05-31 | 山东棉花研究中心 | Pcr identification method for germplasm materials of upland cotton hb red flower |
| CN105154539A (en) * | 2015-08-31 | 2015-12-16 | 中国农业科学院植物保护研究所 | Upland cotton specific PCR (Polymerase Chain Reaction) primer pair and detection method of upland cotton specific primer pair in phytophagous insect body |
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Non-Patent Citations (1)
| Title |
|---|
| 《农业生物技术学报》 20091231 王奕海 一种检测抗虫棉中不同Bt基因表达盒结构的PCR方法 , 第5期 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935705A (en) * | 2010-08-30 | 2011-01-05 | 中国农业科学院植物保护研究所 | PCR detection method of transformant specificity of transgenic insect-resistant cotton Hubei mixed cotton No.1 and application thereof |
| CN101935705B (en) * | 2010-08-30 | 2013-03-06 | 中国农业科学院植物保护研究所 | PCR detection method of transformant specificity of transgenic insect-resistant cotton Hubei mixed cotton No.1 and application thereof |
| WO2012068772A1 (en) * | 2010-11-22 | 2012-05-31 | 山东棉花研究中心 | Pcr identification method for germplasm materials of upland cotton hb red flower |
| CN105154539A (en) * | 2015-08-31 | 2015-12-16 | 中国农业科学院植物保护研究所 | Upland cotton specific PCR (Polymerase Chain Reaction) primer pair and detection method of upland cotton specific primer pair in phytophagous insect body |
| CN105154539B (en) * | 2015-08-31 | 2019-08-20 | 中国农业科学院植物保护研究所 | Upland cotton Specific PCR primers pair and its in the intracorporal detection method of plant-feed insect |
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