CN101413005A - Foreign fragment flanking sequence of transgenic rice line Kefeng No. 8 containing sck/cry1Ac bivalent insect-resistant gene and use - Google Patents
Foreign fragment flanking sequence of transgenic rice line Kefeng No. 8 containing sck/cry1Ac bivalent insect-resistant gene and use Download PDFInfo
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- CN101413005A CN101413005A CNA2008102257240A CN200810225724A CN101413005A CN 101413005 A CN101413005 A CN 101413005A CN A2008102257240 A CNA2008102257240 A CN A2008102257240A CN 200810225724 A CN200810225724 A CN 200810225724A CN 101413005 A CN101413005 A CN 101413005A
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Abstract
The invention relates to a flanking sequence for a foreign inserting fragment of a transgenic rice line Kefeng No. 8 harboring sck/crylAc double insect resistance genes and application thereof, which belong to the field of biotechnology. A flanking sequence of a 5' end is shown in SEQ ID NO1, and a flanking sequence of a 3' end is shown in SEQ ID NO2. The flanking sequence is utilized to design a specific primer for the augmentation of target DNA, which can establish sensitive and specific qualitative and quantificational PCR detection of the line specificity of the rice line Kefeng No. 8.
Description
Technical field
What the present invention relates to is a kind of gene order of biological technical field.Specifically, relate to rich No. 8 external source of a kind of transgenic paddy rice strain section and insert segmental flanking sequence.
Background technology
Paddy rice is one of most important food crop of China, along with broad research and the application of transgenic technology in paddy rice, has had a plurality of transgenic paddy rice strains to carry out environment release, application commercialization plantation.The detection that transgenic plant are carried out strain specificity is to the transgenic plant management that exercises supervision, and ensures the important technology basis of its sound development.And the external source of transgenic plant to insert segmental flanking sequence be one of most important characterization of molecules of transgenic plant strain, therefore, it is the important technology data of setting up transgenic plant strain specificity detection method that external source is inserted segmental flanking sequence.
The transgenic plant exogenous insertion vector flanking sequence that at present partial monopoly and bibliographical information arranged, for example: magnify people such as soldier utilized the TAIL-PCR methods analyst in 2006 the external source of corn strain MON863 insert segmental flanking sequence, set up the strain specificity detection method of transgenosis MON863 corn, people such as Xie Jiajian utilized methods such as TAIL-PCR, genomewalking and LD-PCR to obtain No. 6, transgenic paddy rice Kemingdao, Bt Shanyou 63 and Ke Feng in 2007 external source is inserted segmental flanking sequence, and has set up the detection method of strain specificity.Yet, in analysis, find, also without any the article and the patent report that insert segmental flanking sequence about rich No. 8 external source of transgenic paddy rice strain section to existing patent and document.
Summary of the invention
At the blank in the above-mentioned field, provide rich No. 8 external source of a kind of transgenic paddy rice strain section to insert segmental flanking sequence.
Further, the present invention also provides this transgenic strain specific PCR detection method.
Rich No. 8 external source of a kind of transgenic paddy rice strain section is inserted segmental flanking sequence, and described flanking sequence is 5 ' end flanking sequence, it is characterized in that: 5 ' end flanking sequence is shown in SEQ ID NO1.
Rich No. 8 external source of a kind of transgenic paddy rice strain section is inserted segmental flanking sequence, and described flanking sequence is 3 ' end flanking sequence, it is characterized in that: 3 ' end flanking sequence is shown in SEQ ID NO2.
The preparation method of 5 ' end flanking sequence is characterized in that extracting rich No. 8 plant genome DNAs of transgenic paddy rice strain section, obtains by the TAIL-PCR amplification.
The preparation method of 3 ' end flanking sequence is characterized in that extracting rich No. 8 plant genome DNAs of transgenic paddy rice strain section, obtains by pcr amplification.
Rich No. 8 external source of transgenic paddy rice strain section is inserted the qualitative PCR detection method of segmental flanking sequence, it is characterized in that: two combination of primers in the described PCR reaction are the Auele Specific Primer of 5 ' end flanking sequence: a primer is the forward primer according to 1-1110 bit sequence design among the SEQ ID NO1, and another primer is the reverse primer according to the 1111-1747 bit sequence design of SEQ ID NO1.
Described forward primer is K8P1:5 '-ATATTCTGAAGTGGCCTGTT-3 ',
Described reverse primer is K8P2:5 '-TAATAGCGAAGAGGCCCGCA-3 '.
Rich No. 8 external source of transgenic paddy rice strain section is inserted the qualitative PCR detection method of segmental flanking sequence, it is characterized in that: two combination of primers in the described PCR reaction are the Auele Specific Primer of 3 ' end flanking sequence: the forward primer that primer designs according to 1-583 bit sequence among the SEQ ID NO2, another primer is according to the reverse primer of the 584-650 bit sequence design of SEQ ID NO2.
Described forward sequence is K8P3:5 '-TGCAAGTCCAGGGATGAAGA-3 ',
Described reverse sequence is G3-P1:5 '-TTTATTTGTATAGTGGCGACC-3 '.
Rich No. 8 external source of transgenic paddy rice strain section is inserted segmental flanking sequence qualitative PCR detection method, contains following primer simultaneously in the described PCR reaction,
Forward primer is K8P1:5 '-ATATTCTGAAGTGGCCTGTT-3 ',
Reverse primer is K8P2:5 '-TAATAGCGAAGAGGCCCGCA-3 ';
The forward sequence is K8P3:5 '-TGCAAGTCCAGGGATGAAGA-3 ',
Reverse sequence is G3-P1:5 '-TTTATTTGTATAGTGGCGACC-3 '.
According to Peng Haiying (Peng Haiying. agriculture bacillus mediated marker-free changes the acquisition of divalent insect-resistant gene long-grained nonglutinous rice. the master thesis .2003 of Hua Zhong Agriculture University) the structure (see figure 1) of the carrier pCDMARUBAC-Hyg that is used to transform of report, goal gene that is used to transform (crylAc gene and SCK gene) and selectable marker gene (hpt gene) are positioned at two independently T-DNA zones, and its structure is seen Fig. 1.One of them T-DNA is made up of selectable marker gene hpt expression casette; Another T-DNA is made up of crylAc expression casette and SCK expression casette.
The present invention adopts ordinary method, extracts plant genome DNA, utilizes the TAIL-PCR partition method to obtain 5 ' end flanking sequence 1755bp, and its nucleotide sequence is shown in SEQ ID NO1.By analyzing, this 5 ' end flanking sequence comprises: the rice genome sequence, be positioned at No. 11 karyomit(e)s of paddy rice (the GenBank registration number: 13191855-13192963 position AP008217), identical among its nucleotide sequence and the SEQ ID NO.1 from the nucleotide sequence of 1-1110 position; Conversion carrier pCDMARUBAC-Hyg partial sequence, the nucleotide sequence of 1111-1747 position is identical among its nucleotide sequence and the SEQ ID NO.1;
Utilize pcr amplification to obtain 3 ' end flanking sequence 650bp, its nucleotide sequence is shown in SEQ ID NO2.By analyzing, this 3 ' end flanking sequence comprises: conversion carrier pCUBAC-HYG partial sequence, and the nucleotide sequence of the 1-583 position among its nucleotide sequence and the SEQ ID NO.2 is identical; The rice genome sequence, be positioned at No. 11 karyomit(e)s of paddy rice (the GenBank registration number: 13192994-13193060 position AP008217), identical among its nucleotide sequence and the SEQ ID NO.2 from the nucleotide sequence of 584-650 position.
Rich No. 8 external source of transgenic paddy rice strain section is inserted the qualitative PCR detection method of segmental flanking sequence, holds flanking sequence as the target DNA amplified fragments with 5 ' end flanking sequence and/or 3 '.According to 5 ' end flanking sequence design Auele Specific Primer, its forward primer can be according to the nucleotide sequence design of 1-1110 position, promptly be according to the rice genome sequences Design, be positioned at No. 11 karyomit(e)s of paddy rice (GenBank registration number: 13191855-13192963 position AP008217), reverse primer can be according to the nucleotide sequence design of 1111-1747 position, promptly be to design according to conversion carrier pCDMARUBAC-Hyg partial sequence, the forward primer of the present invention's design is K8P1:5 '-ATATTCTGAAGTGGCCTGTT-3 ', and reverse primer is K8P2:5 '-TAATAGCGAAGAGGCCCGCA-3 '.
According to 3 ' end flanking sequence design Auele Specific Primer, its forward primer can be according to the nucleotide sequence design of 1-583 position, promptly be to design according to conversion carrier pCDMARUBAC-Hyg partial sequence, reverse primer can be according to the nucleotide sequence design of 584-650 position, promptly be according to the rice genome sequence, be positioned at No. 11 karyomit(e)s of paddy rice (the GenBank registration number: 13192994-13193060 position AP008217), the forward sequence of the present invention design is K8P3:
5 '-TGCAAGTCCAGGGATGAAGA-3 ', reverse sequence are G3-P1:
5’-TTTATTTGTATAGTGGCGACC-3’。
The present invention has cloned rich No. 8 external source of transgenic paddy rice strain section first and has inserted segmental flanking sequence, and clear and definite rich No. 8 external source of transgenic paddy rice strain section is inserted segmental flanking sequence and can be used as the target DNA amplified fragments, sets up sensitive, rich No. 8 qualitative, the quantitative PCR detection of strain specificity of specific transgenic paddy rice strain section.The present invention clones, isolating external source is inserted segmental flanking sequence and can be further used for setting up the transgenic strain PCR method for detecting specificity.
Description of drawings
The T-DNA structural representation of Fig. 1 pCDMARUBAC-Hyg,
Rich No. 85 of Fig. 2 section ' end border TAIL-PCR amplification collection of illustrative plates,
M:DNA Marker DL2000, size is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1,3,5,7,9: the TAIL-PCR second stage amplified reaction product of primer AD2～AD6; 2,4,6,8,10: the TAIL-PCR third stage amplified reaction product of primer AD2～AD6
Rich No. 83 of Fig. 3 section ' end flanking sequence pcr amplification collection of illustrative plates,
M:DNAMarker DL2000; 1: combination of primers G3-P1/CptI-p1 amplification,
5 ' end flanking sequence Auele Specific Primer K8P1/K8P2 detection, the M:marker DL2000 that Fig. 4 transgenic paddy rice strain section is rich No. 8; 1,2: rich No. 8 of transgenic paddy rice strain section; 3,4: extensive 86 to throwing light on,
3 ' the end flanking sequence Auele Specific Primer K8P3/G3-P1 detections that Fig. 5 transgenic paddy rice strain section is rich No. 8.M:marker DL2000; 1,2: rich No. 8 of transgenic paddy rice strain section; 3,4: to throwing light on extensive 86.
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, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or the condition of advising according to manufacturer.
The clone of the flanking sequence of the exogenous insertion vector that transgenic paddy rice strain section is rich No. 8
One, experiment material
1, vegetable material
Transgenic paddy rice: rich No. 8 of transgenic paddy rice strain section.
2, enzyme and reagent
Molecular biology reagent, as dNTPs, Taq archaeal dna polymerase, DL2000Marker 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.
3, laboratory apparatus
Pcr amplification instrument: Eppendorf 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.
Two, experimental technique and process
1, oryza sativa genomic dna extracts and detects
1) extraction of oryza sativa genomic dna
Get the seedling stage rice leaf as the material of 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 rice material.
2) oryza sativa genomic dna detects
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, the separation of rich No. 8 flanking sequences of section
1) rich No. 85 ' end flanking sequences of TAIL-PCR separation section
TAIL-PCR be used to the to increase flanking sequence of known region.According to rich No. 8 T-DNA borderline regions design three nido specific PCR primer P1, P2 of section and P3, make up respectively with 5 degenerated primer AD2～AD6 successively and carry out pcr amplification three times, primer sequence sees Table 1.The PCR reaction conditions sees Table 2.
Table 1 is used for the degenerated primer and the Auele Specific Primer of TAIL-PCR amplification
| primers | sequence |
| P1 | 5’-CATCTTCACTCGGTAACATCG-3’ |
| P2 | 5’-GAGTTCATTCCAGTTACTGCAAC-3’ |
| P3 | 5’-GAATCCTGTTGCCGGTCTTG-3’ |
| AD2 | 5’-AGTGNAGAANCAAAGG-3’ |
| AD3 | 5’-CATCGNCNGANACGAA-3’ |
| AD4 | 5’-TG(A/T)GNAG(A/T)ANCA(G/C)AGA-3’ |
| AD5 | 5’-TCGTNCGNACNTAGGA-3’ |
| AD6 | 5’-NTCGA(G/C)T(A/T)T(G/C)G(A/T)GTT-3’ |
Table 2 TAIL-PCR response procedures and condition
2) the rich No. 83 ' separation of end flanking sequence of section
According to the section that has measured rich No. 85 ' end side rice genome sequence, its downstream design primer G3-P1 (sequence is: 5 '-TTTATTTGGATAGTGGCGACC-3 ', the primer in the carrier sequence on the SCK gene (sequence is: CptI-p1:5 '-AAAATGAAGAGCACCATCTTC-3 ') increase by combination.
Reaction system: 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: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations; 72 ℃ of 10min.
3, 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.Adopt the sequence and the carrier sequence similarity of vector NTI10.0 (Invitrogen) comparison and assay determination, in ncbi database (http://www.ncbi.nlm.nih.gov/), retrieve similar rice genome sequence with BLASTN.
Three, experimental result
1,5 ' rich No. 8 end flanking sequence of section
The TAIL-PCR amplification on the rich No. 8 T-DNA borders of section shows, has only the band of second, third grade amplified production of AD5 single in 6 AD primers, and segment size close (Fig. 2).Reclaim the third stage amplified fragments of AD5, link on the pMD18-T carrier, carry out sequencing.
Sequencing is the result show, expanding fragment length is 1747bp, and wherein the 1-1110 position is the rice genome sequence, is positioned at No. 11 karyomit(e)s of paddy rice (GenBank registration number: 13191855-13192963 AP008217); The 1111-1747 position is a conversion carrier pCDMARUBAC-Hyg partial sequence.
2,3 ' rich No. 8 end flanking sequence of section
Utilize G3-P1/CptI-p1 combination pcr amplification to obtain specific amplification, see Fig. 3.Sequencing shows, expanding fragment length is 650bp, wherein the 1-583 position is a conversion carrier pCUBAC-HYG partial sequence, and the 584-650 position is the rice genome sequence, is positioned at No. 11 karyomit(e)s of paddy rice (GenBank registration number: 13192994-13193060 position AP008217).
Insert the qualitative PCR detection method of fragment flanking sequence based on rich No. 8 external source of transgenic paddy rice strain section
One, experiment material
1, vegetable material
Transgenic paddy rice: rich No. 8 of transgenic paddy rice strain section.
Conventional rice: bright extensive 86.
2, enzyme and reagent
Molecular biology reagent, as dNTPs, Taq archaeal dna polymerase, DL2000 Marker 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.
3, laboratory apparatus
Pcr amplification instrument: Eppendorf 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.
Two, experimental technique and process
1, plant genome DNA extracts and detects
See " plant genome DNA extracts and detects " among the embodiment 1
2, the strain specificity PCR based on 5 ' end and 3 ' end flanking sequence detects
According to 5 ' end of measuring among the embodiment 1 and 3 ' end flanking sequence, at its rice genome sequence part and conversion carrier sequence partial design primer, see Table 8 respectively.Pcr amplification is the result show, 5 ' end that transgenic paddy rice strain section is rich No. 8 and 3 ' end amplimer combination have obtained the 207bp of expection and the amplified fragments of 278bp respectively, and all do not have corresponding amplified fragments extensive No. 86 to throwing light on, and see Fig. 4 and Fig. 5.
The rich No. 8 strain specificity PCR of table 8 section detect primer
Attached: sequence table
SEQ ID NO1:5 ' terminal sequence: (1747bp)
1 ACGTAGGATT ACCTAATTGA TTAGAATAGC CTTTGTGGTG GACACTACTT AGAATAGGCT
61 GTGTTCAATA CTATGGGTTG GGAACCCAAA TCCTGTGCAC GGAAAACGGA ACGGTCTATT
121 AGCACGTGAT TAATTAAGTA TTAGCAATTT TTTTTTAAAA ATGGATCAAT ATGATTTTTT
181 TAAGCAACTT TCGTATAAAA ACTTTTTGCA AAAAACACAC CATTTAGCAG TTTGAAAAGT
241 GTGCGCGCGG AATACGAGGG AAAGGGGCTG GGAACCTCCT CATCCGAACG CAGCCTAAGG
301 CTGTGTTCTT CCCCTTCTTT CCCGAGTCAC ATTCTTCTTT TTCTGTGCGC ATGCTTTTGC
361 GAATGTTTTA TATAGGAAAC TTACTTTTAA AAATCATACT AAACCATTTT TCAAGCTCTT
421 TTTTACTAAT ACTTAATTAA TCACGTGTTA ATAGGCAACA CCGTTCCCCG TGTGGGGAAG
481 TGGAGTTACT CCACCTCCTG AACTCAGCCT AATACATCAA AGCTATCATG AGATTACCAA
541 CATGGCTTTT TCTTCGGACT CACAAATAAG ACGGTATTAT AATAACTCAA GGGACAAATC
601 CTTTAATTGA TTCTTTAAAT GATTATGATA AATTCATATA GTAATCTCTA GCGGTGTATA
661 CACTCAAGCA AGACTTAATT CTTTATTTAT TTGAATAATT TTTAATATTG TTTAGTATCT
721 TGGAAATTGA AACGTTGCAT TCGTATTTAA AACTATAGCA TAACAGAATT TTCAATGACT
781 TATACATCCT TAATTTGAAG TATTTCTCTA AATGATACTG GCATATAAAG AGCTAGCTTT
841 GGTTGGAAAC GATAATAAAC TATGCATAGA GGAAATAAAA ATATGTGGGC TTTTACCTTC
901 ACAGTAGCGA ACGTCACATT GTTTCCATAC GTCATGGTGA ATCTGCGAAT CAATTACATT
961 TCAGAGCACA CATGCATAAG CATTATTGTT CAAAATAGTT TGTACCTTTA TATGTATTCT
1021 AAAAATATTC TGAAGTGGCC TGTTATTAAA TGAACTAATA TGCTGAAGCA AATATCTGAC
1081 CCTGCAGACT GGCCATCAAG ATGCCAGGCC TGATAGTTTA AACTGAAGGC GGGAAACGAC
1141 AATCTGATCC AAGCTCAAGC TGCTCTAGCA TTCGCCATTC AGGCTGCGCA ACTGTTGGGA
1201 AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG GATGTGCTGC
1261 AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC
1321 CAGTGCCAAG CTCTGGCGAA AGGGGGATGT GCTGCAAGGC GATTAAGTTG GGTAACGCCA
1381 GGGTTTTCCC AGTCACGGCG TTGTAAAACG ACGGCCAGTG AGCGCGCGTA ATACGACTCA
1441 CTATAGGGCG AATTGGAGCT CCACCGCGGT GGCGGCCGCT CTAGAACTAG TGGATCCCCC
1501 GGGCTGCAGG AATTCGATAT CAAGCTTCTA GAGATCTAGT AACATAGATG ACACCGCGCG
1561 CGATAATTTA TCCTAGTTTG CGCGCTATAT TTTGTTTTCT ATCGCGTATT AAATGTATAA
1621 TTGCGGGACT CTAATCATAA AAACCCATCT CATAAATAAC GTCATGCATT ACATGTTAAT
1681 TATTACATGC TTAACGTAAT TCAACAGAAA TTATATGATA ATCATCGCAA GACCGGCAAC
1741 AGGATTC
SEQ ID NO2:3 ' terminal sequence: (650bp)
1 AAAATGAAGA GCACCATCTT CTTTGCTCTC TTTCTCTTTT GTGCCTTCAC CACCTCATAC
61 CTACCTTCAG CCATCCCTGA TTTCGTTTTA AAGGTGTGTG TGCTGGTACT TTTCCTTGTA
121 GGGGTTACTA CTGCAGCCAT GGATCTGAAC CACCTCGGAA GTAATCATCA TGATGACTCA
181 AGCGATGAAC CTTCTGAGTC TTCAGAACCA TGCTGCGATT CATGCATCTG CACTAAATCA
241 ATACCTCCTC AATGCCATTG TACAGATATC AGGTTGAATT CGTGTCACTC GGCTTGCAAA
301 TCCTGCATGT GTACACGATC AATGCCAGGC AAGTGTCGTT GCCTTGACGT TGCTGATTTC
361 TGTTACAAAC CTTGCAAGTC CAGGGATGAA GATGATGAGA AAGATGAACT CTAGATCAAG
421 CTCGATTCTA GAGTCAAGCA GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGA
481 ATCCTTTGC CGGTCTTGCG ATGATTATCA TATAATTTCT GTTGAATTAC GTTAAGCATG
541 TAATAATTAA CATGTAATGC ATGACGTTAT TTATGAGATG GGTGATCTCA CCCATGCTTG
601 GGTACTAAGA AATGGCAGAA CAGCATCATG GTCGCCACTA TACAAATAAA
SEQ ID NO3：
K8P1：5’-ATATTCTGAAGTGGCCTGTT-3’
SEQ ID NO4：
K8P2：5’-TAATAGCGAAGAGGCCCGCA-3’
SEQ ID NO5：
K8P3：5’-TGCAAGTCCAGGGATGAAGA-3’
SEQ ID NO6：
G3-P1：5’-TTTATTTGTATAGTGGCGACC-3’
Claims (9)
1. rich No. 8 external source of transgenic paddy rice strain section is inserted segmental flanking sequence, and described flanking sequence is 5 ' end flanking sequence, it is characterized in that: 5 ' end flanking sequence is shown in SEQ ID NO1.
2. rich No. 8 external source of transgenic paddy rice strain section is inserted segmental flanking sequence, and described flanking sequence is 3 ' end flanking sequence, it is characterized in that: 3 ' end flanking sequence is shown in SEQ ID NO2.
3. the preparation method of the described flanking sequence of claim 1 is characterized in that extracting rich No. 8 plant genome DNAs of transgenic paddy rice strain section, obtains by the TAIL-PCR amplification.
4. the preparation method of the described flanking sequence of claim 2 is characterized in that extracting rich No. 8 plant genome DNAs of transgenic paddy rice strain section, obtains by pcr amplification.
5. rich No. 8 external source of transgenic paddy rice strain section is inserted segmental flanking sequence qualitative PCR detection method, it is characterized in that: two Auele Specific Primers that combination of primers is the described flanking sequence of claim 1 in the described PCR reaction: a primer is the forward primer according to 1-1110 bit sequence design among the SEQID NO1, and another primer is the reverse primer according to the 1111-1747 bit sequence design of SEQ ID NO1.
6. detection method according to claim 5,
Described forward primer is K8P1:5 '-ATATTCTGAAGTGGCCTGTT-3 ',
Described reverse primer is K8P2:5 '-TAATAGCGAAGAGGCCCGCA-3 '.
7. rich No. 8 external source of transgenic paddy rice strain section is inserted segmental flanking sequence qualitative PCR detection method, two Auele Specific Primers that combination of primers is the described flanking sequence of claim 2 in the described PCR reaction: the forward primer that primer designs according to 1-583 bit sequence among the SEQ ID NO2, another primer is according to the reverse primer of the 584-650 bit sequence design of SEQ ID NO2.
8. detection method according to claim 5,
Described forward sequence is K8P3:5 '-TGCAAGTCCAGGGATGAAGA-3 ',
Described reverse sequence is G3-P1:5 '-TTTATTTGTATAGTGGCGACC-3 '.
9. rich No. 8 external source of transgenic paddy rice strain section is inserted segmental flanking sequence qualitative PCR detection method, contains following primer simultaneously in the described PCR reaction,
Forward primer is K8P1:5 '-ATATTCTGAAGTGGCCTGTT-3 ',
Reverse primer is K8P2:5 '-TAATAGCGAAGAGGCCCGCA-3 ';
The forward sequence is K8P3:5 '-TGCAAGTCCAGGGATGAAGA-3 ',
Reverse sequence is G3-P1:5 '-TTTATTTGTATAGTGGCGACC-3 '.
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| CN103014159A (en) * | 2012-12-07 | 2013-04-03 | 南京农业大学 | Molecular detection method for Cry1Ac toxin non-recessive resistance cadherin cytomere deletion mutation of cotton bollworm |
| CN103014159B (en) * | 2012-12-07 | 2014-07-02 | 南京农业大学 | Molecular detection method for Cry1Ac toxin non-recessive resistance cadherin cytomere deletion mutation of cotton bollworm |
| CN103060459A (en) * | 2013-01-17 | 2013-04-24 | 中国检验检疫科学研究院 | Primer, probe, kit and method for detecting #8 Kefeng transgenic rice strain |
| CN103834645A (en) * | 2014-03-06 | 2014-06-04 | 中国农业科学院生物技术研究所 | Connecting region sequence of exogenous inserting gene and flanking rice sequence of transgenic rice kefeng No.2 and application thereof |
| CN103834736A (en) * | 2014-03-06 | 2014-06-04 | 中国农业科学院生物技术研究所 | PCR (Polymerase Chain Reaction) detection primer in homozygous/heterozygous state of exogenous gene of transgenic rice Kefeng No.2, detection method and kit |
| CN103834736B (en) * | 2014-03-06 | 2016-03-23 | 中国农业科学院生物技术研究所 | The PCR of the rich 2 extra source gene pure/heterozygous states of transgenic paddy rice section detects primer, detection method and test kit |
| CN103834645B (en) * | 2014-03-06 | 2016-07-13 | 中国农业科学院生物技术研究所 | Transgenic paddy rice section inserts gene and the bonding pad sequence of flank rice sequences and application in rich 2 extra sources |
| CN104388421A (en) * | 2014-08-28 | 2015-03-04 | 上海市农业科学院 | Exogenous insert flanking sequence of transgenic rice strain 134Bt, amplification primer and application thereof |
| CN110724704A (en) * | 2019-10-16 | 2020-01-24 | 安徽省农业科学院水稻研究所 | Positive plasmid for identifying transgenic rice transformant and construction method and application thereof |
| CN110863062A (en) * | 2019-11-26 | 2020-03-06 | 扬州大学 | Detection method of anti-sheath blight rice line with OsPGIP1 and GAFP2 transgenic bivalent genes |
| CN110872635A (en) * | 2019-12-06 | 2020-03-10 | 扬州大学 | Detection method of transgenic bivalent sheath blight-resistant rice strain WYJ24-PG-10-1 |
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