CN101392258B - PCR method for identification of nucotn 33B and GK12 and dependent Bt gene expression cassettes - Google Patents
PCR method for identification of nucotn 33B and GK12 and dependent Bt gene expression cassettes Download PDFInfo
- Publication number
- CN101392258B CN101392258B CN2008102257255A CN200810225725A CN101392258B CN 101392258 B CN101392258 B CN 101392258B CN 2008102257255 A CN2008102257255 A CN 2008102257255A CN 200810225725 A CN200810225725 A CN 200810225725A CN 101392258 B CN101392258 B CN 101392258B
- Authority
- CN
- China
- Prior art keywords
- cotton
- seq
- sequence
- pcr method
- gene expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to 'a PCR method for identifying transgenic pest-resistance cotton of new cotton 33B and GK12 and Bt gene expression frame relied on by the method', pertaining to the biological technology field. The sequence of Bt gene expression frame of the transgenic pest-resistance cotton of new cotton 33B is shown as Seq ID No.1, and the sequence of Bt gene expression frame of the transgenic pest-resistance cotton of new cotton GK12 is shown as Seq ID No.2; the PCR method for identifying new cotton 33B and GK12 comprises a specificity detection primer designed by utilizing the different sequences of Seq ID No.1 and Seq ID No.2. The PCR method can utilize general PCR instruments and reagents to identify the new cotton 33B and GK12 and cotton strain with the new cotton 33B or/andGK12 as seed-parent rapidly and accurately.
Description
Technical field
The invention belongs to biological technical field.Particularly relate to a kind of PCR method of new cotton 33B of transgenic cotton against pests and GK12 and Bt gene expression frame of dependence thereof differentiated.
Background technology
Since the tomato Flavr of first commercialization transgenosis Cultivar-delayed maturity Savr
TMSince the birth, the 13rd year of human lives stepped in the commercialization of genetically modified crops plantation.At present, the country of whole world plantation genetically modified crops has 23, and cultivated area adds up 1.143 hundred million hectares.China is one of main plantation country of genetically modified crops, and the transgenic plant of having ratified the commercialization plantation comprise the tomato of transgenic pest-resistant cotton and willow, the transgenic papaya of anti-pawpaw ring spot virus, antiviral capsicum and delayed maturity etc.Wherein, trans Bt gene (Bacillus thuringiensis) Insect Resistant Cotton is the genetically modified crops of China's cultivated area maximum, and cultivated area reached 3,800,000 hectares in 2007, accounted for 69% of Cotton in China total cultivated area.In the transgenic cotton against pests that China extensively plants at present, with the anti-series of state of the Mon531 transformant of Monsanto company (as new cotton 33B etc.) and Biological Technology institute, Chinese Academy of Agricultural Sciences's development (as GK-12 etc.) is that two big important masters plant strain, set up a kind of method for quick of differentiation two big transgene cotton strains, not only can provide technical support for the detection of two strains, simultaneously to understanding the national distribution situation of two strain transgene cottons, the distinct shared separately market share all has important practical application meaning.
(Yang et al such as Yang, 2005, Transgenic Research, 14:817-831) the foreign DNA integrated structure (Fig. 1) of Bao Dao transgenic cotton against pests MON531, wherein the expression cassette of Bt gene (crylAc gene) is made up of 35S promoter, crylAc gene and 7S terminator, but does not report concrete sequence information.In analysis, find, also without any about the Bt gene expression frame sequence of new cotton 33B of transgenic cotton against pests and GK12 with to the article and the patent report of its discrimination method existing patent and document.
Summary of the invention
At the blank in the above-mentioned field, the Bt gene expression frame sequence of new cotton 33B of transgenic cotton against pests and GK12 and the PCR method of differentiating these two kinds of transgenic cotton flower varieties are provided, this PCR method can utilize common PCR instrument, reagent quick and precisely to differentiate make new advances cotton 33B and GK12, and with new cotton 33B or/and GK12 is parent's a cotton strain.
The Bt gene expression frame sequence of the new cotton 33B of transgenic cotton against pests, shown in Seq ID No.1, wherein the sequence of promotor is positioned at 1-277bp, and the gene order of foreign gene cry1Ac is positioned at 278-3811bp, and the sequence of terminator is positioned at 3812-4074bp.
The Bt gene expression frame sequence of transgenic pest-resistant cotton GK 12, shown in Seq ID No.2, wherein the sequence of promotor is positioned at 1-277bp, and the gene order of foreign gene crylAc is positioned at 278-2197bp, and the sequence of terminator is positioned at 2198-2471bp.
A kind of PCR method of differentiating new cotton 33B and GK12, comprise forward primer 33B-P or/and forward primer GK12-P in the described PCR system,, a reverse primer MG-P, it is characterized in that the 278bp-3811bp position of 33B-P design at Seq ID No.1, the GK12-P design is in the 278bp-2197bp position of Seq ID No.2, and reverse primer MG-P designs on the back 257bp of Seq ID No.1 or Seq IDNo.2; The position of described two forward primers design should make two kinds of differences in length that detect product more than 50bp.
Described 33B-P design is in the 2198bp-3782bp position of Seq ID No.1.
Described 33B-P design is in the 3765bp-3782bp position of Seq ID No.1; Described GK12-P design is in the 2016bp-2036bp position of Seq ID No.2; Described MG-P design is in the 4055bp-4074bp position of Seq ID No.1.
The extension time is 30 seconds in the reaction conditions of described PCR method.
Being applied to of above-mentioned PCR method identifies that with new cotton 33B/ or GK12 be parent's cotton strain.
The present invention is based on MON531 is the parent of new cotton 33B, according to (Yang et al such as Yang, 2005, TransgenicResearch, 14:817-831) Bao Dao MON531 foreign DNA integrated structure, the present invention is respectively at 35S promoter, crylAc gene and 7S3 ' terminator zone design primer, adopt PCR and long-chain pcr amplification to go out to comprise 5 ' terminal sequences of promoter region, and the 3 ' terminal sequences that comprise the terminator zone, through the exogenous Bt expression of gene frame sequence of the new cotton 33B of splicing acquisition, its length is 4074bp, and its nucleotide sequence is shown in Seq ID No.1, draw by sequential analysis, the 1-277bp position of this sequence is a promoter region; The 278-3811bp position is that foreign gene is Bt gene fragment zone; The 3812-4074bp position is 7S terminator zone.And obtain the Bt gene expression frame sequence of GK12 with same method, and its length is 2471bp, shown in Seq ID No.2, draws by sequential analysis, the 1-277bp position of this sequence is a promoter region; The 278-2197bp position is that foreign gene is Bt gene fragment zone; The 2198-2471bp position is 7S terminator zone.
More than the 50bp, promptly the amplified production length of combination of primers 33B-P/MG-P and GK12-P/MG-P amplified production length differ more than the 50bp.Method of the present invention has remedied blank for the detection method of present new cotton 33B and GK12, can identify the purpose transformed variety rapidly and accurately, the whole nation that is a kind of quick understanding two strain transgene cottons distributes and applicable cases, the effective means of the distinct shared separately market share.
Method of the present invention mainly depends on round pcr, in order to improve detection efficiency and accuracy, is the 2198-3811bp position of Seq ID No..1 with forward primer 33B-P design in the difference zone of Seq ID No.1 and Seq ID No.2, more preferably with the 3765bp-3782bp position of 33B-P design at Seq ID No.1, sequence shown in Seq ID No.3, called after 33B-P
1And GK12-P design is in the 2016bp-2036bp position of Seq ID No.2, sequence shown in Seq ID No.4, called after GK12-P
1Reverse primer MG-P design is in the 4055bp-4074bp position of Seq ID No.1, sequence shown in Seq ID No.5, called after MG-P
1, the detection product of the new cotton 33B that preferred primer increases out and the detection product length of GK12 are respectively 310bp, 456bp, can separate fast on agarose gel.So on the one hand because detection product sequence is shorter, requirement for PCR instrument and reagent can be very not high yet, make PCR method of the present invention be easier to apply, on the other hand, the specificity and the accuracy of this detection method had both been improved, because of the distance that has shortened between forward primer and the reverse primer, the extension time of the PCR program that adopts when detecting can be shortened a lot, thereby improved detection efficiency again.In the PCR detection method of the present invention, the PCR program extension time is to get final product in 30 seconds.
New cotton 33B provided by the invention, the Bt gene expression frame sequence of GK12 and the PCR method that relies on its foundation can be used as the new cotton 33B of a kind of evaluation, GK12, and with the effective means of these two kinds transgenic cotton strain that is the parent, because in the transgenic cotton against pests that China extensively plants at present, with the anti-series of state of the Mon531 transformant of Monsanto company (as new cotton 33B etc.) and Biological Technology institute, Chinese Academy of Agricultural Sciences's development (as GK-12 etc.) is two big main cultivars, therefore the present invention is the national distribution situation of distinguishing two big transgene cotton strains and investigating two strain transgene cottons, the distinct shared separately market share, provide convenience, 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 foreign DNA integrated structure of Fig. 1 transgenic cotton against pests MON531
Fig. 2 Bt gene expression frame promotor end fragment augmentation detection electrophorogram
M:Marker DL2000 wherein; 1,2:GK12; 3,4:33B; 5: blank
Fig. 3 Bt gene expression frame terminator end fragment augmentation detection electrophorogram
M:Marker λ/HindIII wherein; 1,2:GK12; 3,4:33B; 5: blank
Fig. 4 PCR method of the present invention is differentiated new cotton 33B and GK12.
M:MarkerDL2000 wherein; 1-10,21:GK12; 11-20,22:33B; 23: blank
Fig. 5 PCR method of the present invention is differentiated the checking of new cotton 33B and GK12
M:Marker DL2000 wherein; N: negative control; B: blank; The heterozygote of P:33B and GK12; 1-20: differentiate sample, swimming lane 1-6,8-12,15,17 and 20 are 33B; Swimming lane 13,14 is GK12; Swimming lane 7,18 is the heterozygosis of 33B and GK12; Swimming lane 16,19 is not 33B or GK12.
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 the Bt gene expression frame sequence of new cotton 33B of transgenic cotton against pests and GK12
1 experiment material
1.1 vegetable material
Transgenic cotton against pests: new cotton 33B and GK12.
1.2 enzyme and reagent
Molecular biology reagent, as dNTPs, Taq archaeal dna polymerase, DL2000 Marker, λ/HindIII Marker 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.
2.1.4 DNA detection
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.
2.2 the acquisition of the Bt gene expression frame promotor terminal sequence of new cotton 33B and GK12
With reference to " No. 953 bulletin-6-2007 transgenic plant of the Ministry of Agriculture and products thereof composition detection insect-proof rice qualitative PCR method ", choose the forward primer 35S-F1 of CaMV35S promotor and the reverse primer Bt-R1 of Bt gene, primer sequence is as shown in table 1, the Bt gene expression frame promotor terminal sequence of be used to increase new cotton 33B of transgenic cotton against pests and GK12.
Reaction system: 0.25 μ L rTaq (5U/ μ L), 5 μ L10 * 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: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations; 72 ℃ of 10min.
2.3 the acquisition of the Bt gene expression frame terminator terminal sequence of new cotton 33B of Insect Resistant Cotton and GK12
Adopt the Bt gene expression frame terminator terminal sequence of LD-PCR method amplification new cotton 33B of transgenic cotton against pests and GK12 in the present embodiment.With reference to the detection primer of Bt gene in " No. 953 bulletin-6-2007 transgenic plant of the Ministry of Agriculture and products thereof composition detection insect-proof rice qualitative PCR method ", choose its forward primer (primer sequence sees Table 1); From ncbi database (http://www.ncbi.nlm.nih.gov/), download 7S3 ' terminator sequence (accession number is J01293), and design primer MG-P1 is as reverse primer, shown in Seq ID No.5 or table 1.
Table 1 is used for the primer of PCR and LD-PCR amplification
| Primer | Sequence |
| 35S-F1 | 5’-GCTCCTACAAATGCCATCATTGC-3’ |
| Bt-F1 | 5’-GAAGGTTTGAGCAATCTCTAC-3’ |
| Bt-R1 | 5’-CGATCAGCCTAGTAAGGTCGT-3’ |
| MG- |
5′-ATACGTGCCAAGTGCCAACC-3’ |
The LD-PCR amplification system is: total system 50 μ L, Ex Taq (Takara co., Dalian), 1.25U 10 * Ex Taq Buffer5 μ L, dNTP (each 2.5mM) 4 μ L, paddy DNA 2 μ L (10ng/ μ L), each 2 μ L (25 μ M) of primer.
LD-PCR amplification program: 94 ℃ of 4min; (98 ℃ of 10s, 68 ℃ of 15min), 30 cycles; 72 ℃ of 10min.
2.4 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 1300 Genetic Analyzer to carry out sequencing.Employing software vector NTI10.0 (Invitrogen) splices the sequence of measuring and analyzes.
3 experimental results
3.1 the sequence of the Bt gene expression frame of the new cotton 33B of transgenic cotton against pests
The Bt gene promoter end and the terminator terminal sequence length that adopt PCR and LD-PCR method to obtain the new cotton 33B of transgenic cotton against pests respectively are respectively 878bp and 3497bp, see Fig. 2 and Fig. 3.Obtained the sequence of the Bt gene expression frame of the new cotton 33B of transgenic cotton against pests through splicing, its length is 4074bp, and its nucleotide sequence is shown in Seq ID No.1.Draw by sequential analysis, the 1-277bp position of this sequence is a promoter region; The 278-3811bp position is that foreign gene is Bt gene fragment zone; The 3812-4074bp position is 7S terminator zone.
3.2 the sequence of the Bt gene expression frame of transgenic pest-resistant cotton GK 12
The Bt gene promoter end and the terminator terminal sequence length that adopt PCR and LD-PCR method to obtain transgenic pest-resistant cotton GK 12 respectively are respectively 878bp and 1894bp, see Fig. 2 and Fig. 3.Obtained the sequence of the Bt gene expression frame of transgenic pest-resistant cotton GK 12 through splicing, its length is 2471bp, and its nucleotide sequence is shown in Seq ID No.2.Draw by sequential analysis, the 1-277bp position of this sequence is a promoter region; The 278-2197bp position is that foreign gene is Bt gene fragment zone; The 2198-2471bp position is 7S terminator zone.
3.3 the comparison of the Bt gene expression frame sequence of new cotton 33B of transgenic cotton against pests and GK12
Bt gene expression frame sequence to new cotton 33B of transgenic cotton against pests and GK12 compares, find that two sequences is on all four at preceding 2130bp and back 257bp, only the 2131-2214 position in the 2131-3817 position of the Bt of transgenic cotton against pests 33B gene expression frame sequence (Seq ID No.1) and the Bt gene expression frame sequence (Seq ID No.2) of transgenic pest-resistant cotton GK 12 is different.
PCR method of the present invention is applied to differentiate PCR experiment and the checking of new cotton 33B of transgenic cotton against pests and GK12
1 experiment material
1.1 vegetable material
Transgenic cotton against pests: new cotton 33B and GK12.
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 PCR method experiment and the checking of new cotton 33B of transgenic cotton against pests and GK12
PCR method of the present invention is differentiated new cotton 33B experiment: according to the sequence Seq ID No.1 that measures among the embodiment 1, at the 3765-3782bp bit sequence design forward primer 33B-P of the Bt of new cotton 33B gene expression frame sequence shown in Seq ID No.1
1, shown in Seq ID No.3, at the reverse primer MG-P of 4055-4074bp position sequence design
1, shown in Seq ID No.5; Adopt primer 33B-P
1/ MG-P
1Combination, 10 individual plants of the new cotton 33B of picked at random transgenic cotton against pests are that template is carried out pcr amplification, the result all can obtain the specific amplified fragment of 310bp, see Fig. 4, the explanation of this experimental result the present invention conform to the Bt gene expression frame sequence of new cotton 33B by the Bt gene expression frame sequence Seq ID No.1 that the amplification splicing is obtained.
PCR method of the present invention is differentiated the GK12 experiment: according to the sequence Seq ID No.2 that measures among the embodiment 1, at the 2016-2036 bit sequence design forward primer GK12-P of the Bt of transgenic pest-resistant cotton GK 12 gene expression frame sequence shown in Seq ID No.1
1, shown in Seq ID No.4, at the reverse primer MG-P of 2452-2471 bit sequence design
1, shown in Seq ID No.5; Adopt primer GK12-P
1/ MG-P
1Combination, 10 individual plants of picked at random transgenic pest-resistant cotton GK 12 carry out pcr amplification, the result all can obtain the specific amplified fragment of 456bp, see Fig. 4, the explanation of this experimental result the present invention conform to the Bt gene expression frame sequence of GK12 by the Bt gene expression frame sequence Seq ID No.2 that the amplification splicing is obtained.
PCR method of the present invention is applied to differentiate the checking of new cotton 33B and GK12 and other kinds: adopt primer 33B-P1/MG-P1/GK12-P1 combination, detect 20 cotton samples available from market.See Fig. 5.Laboratory test results shows that PCR method provided by the invention can identify 33B, GK12 and both heterozygotes well, and gets rid of other kinds.
Above 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, 55 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 35 circulations; 72 ℃ of 10min.
Table 2 is differentiated the PCR primer of new cotton 33B of transgenic cotton against pests and GK12
| Primer | Sequence |
| 33B-P1 | 5’-AGGGAACCTTCATCGTGG-3’ |
| GK12-P1 | 5’-CATCTTCACTCGGTAACATCG-3’ |
| MG-P1 | 5’-ATACGTGCCAAGTGCCAACC-3’ |
Appendix: sequence table
Seq ID No.1: the Bt gene expression frame sequence of new cotton 33B: (4074bp)
The Bt gene expression frame sequence of Seq ID No.2:GK12: (2471bp)
Seq ID No.3 forward primer 33B-P
1
Seq ID No.4 forward primer GK12-P
1
Seq ID No.5 reverse primer MGB-P
1
Seq ID No.6 35S promoter forward primer 35S-F1
Seq ID No.7Bt gene forward primer Bt-F1
Seq ID No.8Bt gene reverse primer Bt-R1
Claims (5)
1. PCR method of differentiating new cotton 33B and GK12, comprise forward primer that detects new cotton 33B and the forward primer that detects GK12 in the described PCR system,, a reverse primer, it is characterized in that detecting the 278bp-3811bp position of the forward primer design of new cotton 33B at Seq ID No.1, the forward primer design that detects GK12 is in the 278bp-2197bp position of Seq ID No.2, and reverse primer designs on the back 257bp of Seq ID No.1 or Seq ID No.2; The position of described two forward primers design should make two kinds of differences in length that detect product more than 50bp.
2. PCR method according to claim 1, the forward primer design of the new cotton 33B of described detection is in the 2198bp-3782bp position of Seq ID No.1.
3. PCR method according to claim 2, the forward primer design of the new cotton 33B that detects is in the 3765bp-3782bp position of Seq ID No.1; The forward primer design of described detection GK12 is in the 2016bp-2036bp position of Seq ID No.2; Described reverse primer design is in the 4055bp-4074bp position of Seq ID No.1.
4. PCR method according to claim 3, the extension time is 30 seconds in the reaction conditions of described PCR method.
5. PCR method according to claim 1, described new cotton 33B and GK12 comprise that also with new cotton 33B and GK12 be parent's cotton strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102257255A CN101392258B (en) | 2008-11-10 | 2008-11-10 | PCR method for identification of nucotn 33B and GK12 and dependent Bt gene expression cassettes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102257255A CN101392258B (en) | 2008-11-10 | 2008-11-10 | PCR method for identification of nucotn 33B and GK12 and dependent Bt gene expression cassettes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101392258A CN101392258A (en) | 2009-03-25 |
| CN101392258B true CN101392258B (en) | 2010-12-08 |
Family
ID=40492769
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102257255A Expired - Fee Related CN101392258B (en) | 2008-11-10 | 2008-11-10 | PCR method for identification of nucotn 33B and GK12 and dependent Bt gene expression cassettes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101392258B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792810B (en) * | 2010-04-07 | 2012-06-06 | 中国农业科学院植物保护研究所 | PCR ( Polymerase Chain Reaction) method for identifying crylAa gene expression frame in transgenosis pest-resistant cotton |
| CN102719524A (en) * | 2010-07-23 | 2012-10-10 | 苏州大学 | Kit for warfarin personalized medication related gene SNP locus detection and PCR amplification method using same |
| CN103525834A (en) * | 2013-04-25 | 2014-01-22 | 湖南农业大学 | Crylac gene and application thereof |
| CN106676171B (en) * | 2016-11-23 | 2021-04-09 | 中国农业科学院棉花研究所 | Molecular detection method for cotton polygene pyramiding breeding |
-
2008
- 2008-11-10 CN CN2008102257255A patent/CN101392258B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101392258A (en) | 2009-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101240278B (en) | Side sequence of exogenous insert of transgene paddy strain Kemingdao 1 | |
| CN101413005B (en) | Foreign fragment flanking sequence of transgenic rice line Kefeng No. 8 containing sck/cry1Ac bivalent insect-resistant gene and use | |
| CN101392258B (en) | PCR method for identification of nucotn 33B and GK12 and dependent Bt gene expression cassettes | |
| CN103468790B (en) | Primers used for detecting polymorphism of saccharum arundinaceum, and detection method and applications | |
| CN101974618B (en) | Transformant specific polymerase chain reaction (PCR) detection method for transgenic rice strain T1c-19 with cry1C gene | |
| CN101880725B (en) | Polymerase chain reaction (PCR) detection method for trans-cry2A-genic rice line T2A-1 and application thereof | |
| CN111996282B (en) | SSR marker CH0211 for identifying soybean mosaic virus resistant SC3 strain of soybean as well as detection method and application thereof | |
| CN102586306A (en) | Standard plasmid molecules for transgenetic soybean BPS-CV127-9 detection, constructing method thereof and application thereof | |
| CN102888398A (en) | Flanking sequence of exogenous insertion fragment of transgenic rice variety Bar68-1 and application thereof | |
| CN103525810A (en) | Exogenous gene insertion site flanking sequence of phytase transgenic corn and detection method | |
| CN101792814B (en) | PCR method for detecting specificity of transformant of transgenic pest-resistant cotton GK12 | |
| CN114410802B (en) | RPA primer, probe, kit and application for detecting heterodera avenae wollenweber | |
| CN114507743B (en) | RPA primer, probe and kit for rapidly detecting heterodera filipjevi and application of RPA primer, probe and kit | |
| CN102409082A (en) | Five-gene standard plasmid molecule for transgenic soybean detection and construction thereof | |
| CN106701968B (en) | Primer for simultaneously detecting botryococcus and application thereof | |
| CN114214448A (en) | SNP marker for identifying brown planthopper resistant gene Bph30 of rice and application thereof | |
| CN101792810B (en) | PCR ( Polymerase Chain Reaction) method for identifying crylAa gene expression frame in transgenosis pest-resistant cotton | |
| CN107267503A (en) | Method and kit of nucleic acid and detection transgenic paddy rice B1C893 and its Derivative line and application thereof | |
| CN101792812B (en) | PCR ( Polymerase Chain Reaction) method for identifying transformant specificity of phytase gene corn BVLA430101 | |
| RU2265668C1 (en) | Set of primers for detection and/or identification of transgene dna sequences in vegetable material and product comprising thereof (variants), primer (variants), pair of primers (variants), method for detection and/or identification with their using (variants) and device for realization of method | |
| CN105039526B (en) | Method based on the lucky raw round-grained rice 2 of foreign gene flanking sequence identification transgenic paddy rice | |
| CN101792813B (en) | PCR (Polymerase Chain Reaction) method for identifying transistance phytocide cotton LLCOTTON 25 transformant | |
| CN109628632A (en) | A kind of primer combination, probe, kit and method for transgenic corns MON87419 event-specific detection | |
| CN101792811B (en) | PCR ( Polymerase Chain Reaction) method for identifying phytase gene expression frame construction in transgenosis corn | |
| CN104357576B (en) | A kind of Specific primer pair identifying rice varieties fine horse excellent 522 and parent thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20101208 Termination date: 20181110 |