CN102344964A - Detection method for genetically modified wheat B72-8-11 b - Google Patents
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Abstract
The invention relates to a detection method for genetically modified wheat B72-8-11 b, namely detecting a genetic component of which the sequence is SEQ ID NO: 11 by a PCR (polymerase chain reaction) method. The genetic component comprises an edge sequence of which the sequence is SEQ ID NO: 14. By the method, variety identification of the genetically modified wheat B72-8-11 b can be quickly and sensitively realized. The method has high specificity and high sensitivity and is fast and accurate.
Description
Technical field
The present invention relates to a kind of authentication method of transgenic wheat strain.
Background technology
Transgenic wheat is to utilize genetic engineering technique, in the exogenous genes introduced into wheat gene, through screening the wheat that can express goal gene that obtains.In transgenosis and expression study, need carry out Molecular Detection to seedling, to eliminate non-transformed plant through conversion processing.Selectable marker gene (like neomycin phosphotransferase gene, chloramphenicol acetyl transferasegene and Totomycin transferase gene etc.) and reporter gene (like B2 glucosiduronatase gene and luciferase gene etc.) have been widely used in the screening of transformed plant and tissue; But in some cases, this screening effect is unreliable.PCR is used for one of common technology that this purpose detects, and has sensitivity, advantage fast.Be to seek mark property gene fragment appropriate, high specificity for this The Application of Technology, and design primer and the amplification condition that is complementary based on this.
Summary of the invention
The object of the present invention is to provide the method that to identify transgenic wheat B72-8-11b quickly and accurately; The detection method of described transgenic wheat B72-8-11b; Be method through PCR to detect sequence be the gene assembly of SEQ ID NO:11, comprise in this gene assembly that sequence is the edge sequence of SEQ ID NO:14.
The primer of the pcr amplification that the method for the invention described above is addressed is to being SEQ ID NOS:1/2:
B72-F1(SEQ?ID?NO：1)：ATGATGGAGGGCATTACTTGGGAGTGAA
B72-R1(SEQ?ID?NO：2)：ACCCTGATCCCCAAAGTAAACTCGC。
Understand easily, adopt aforesaid method to carry out pcr amplification, if amplification is positive, wheat then to be measured is transgenic wheat B72-8-11b; If amplification is negative, wheat then to be measured is not transgenic wheat B72-8-11b.
More in the optimized technical scheme, the PCR reaction conditions is
94 ℃, 1min, 1 circulation;
98 ℃ of 10s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations;
72 ℃, 7min, 1 circulation.
The optimal technical solution of the detection method of transgenic wheat B72-8-11b of the present invention is:
Adopt 50 μ LPCR reaction systems, wherein contain: testing sample DNA, the preferred 5ng/ μ of its concentration L, 10 * LA PCR BufferII (Mg
2+Plus) 5ul, each 0.4mM of dNTP, base sequence are each 0.2 μ M of PCR primer of SEQ ID NOS:1～2, Taq archaeal dna polymerase 0.05U/ul.
Adopt method of the present invention can realize fast, delicately the strain of transgenic wheat B72-8-11b is identified, this method high specificity, highly sensitive, quick and precisely.
Description of drawings
Accompanying drawing 3 width of cloth of the present invention,
Fig. 1 is that embodiment 1 is 1% agarose gel electrophoresis figure of template amplification with sample C genomic dna, and wherein: M is DL2,000 DNA Marker; 1～6 is respectively ubiquitin, NOS, bar1, bar2, uidA and GAG56D.
Fig. 2 is that embodiment 1 is 1% agarose gel electrophoresis figure of template amplification with sample A, B, C genomic dna; Wherein: M1 is λ-Hind III digest; M2 is DL2; 000 DNA Marker; 1～6 is respectively C-ubiquitin F/Nos R, C-ubiquitin F/bar R1, C-ubiquitin F/uidA R; A-ubiquitin F/Nos R, A-ubiquitin F/bar R1 and A-ubiquitin F/uidA R.
Fig. 3 is the 1% agarose gel electrophoresis figure of embodiment 1, M:DL2 wherein, 000 DNA Marker.
Embodiment
The present inventor successfully clones the gene assembly A that comprises specificity exogenous array among the transgenic wheat B72-8-11b through big quantity research, comprises uidA among this gene assembly A, ubi, lacZ 1Dx5 and wheat cdna group partial sequence.The complete sequence of this gene assembly A is shown in SEQ ID NO:11.On the basis of the said gene assembly A complete sequence that is obtained, the contriver further obtains the edge sequence of transgene component, i.e. sequence shown in the SEQ ID NO:14, and it is positioned at the 8213rd～10384 of SEQ ID NO:11 sequence.Detection to the gene assembly shown in the SEQ ID NO:11 can realize through PCR; Use specific primer that SEQ ID NOS:1/2 is increased under certain condition; As amplify target sequence, promptly decidable institute test sample is derived from transgenic wheat B72-8-11b.
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.
Obtaining of embodiment 1. transgenic wheat specific gene assembly A, B, C complete sequence and respective edges sequence
1, transgenosis sample gene group DNA extraction
Choose transgenic wheat B72-8-11b, transgenic wheat B73-6-1 and transgenic wheat B102-1-2 respectively, be labeled as sample A, sample B and sample C respectively.Each sample is pulverized back pulverization in liquid nitrogen, uses DNA Extraction Kit for GMO Detection Ver.2.0 (TaKaRa Code No.D9093) to carry out extracting genome DNA then.
Detect the DNA sample quality with 1% agarose gel electrophoresis; And use ultramicrospectrophotometer (Nanodrop) to test sample genomic dna quality and concentration; Detected result shows: the genomic dna quality meets the subsequent experimental requirement; The average genomic dna concentration of sample A, B and C is 162.69ng/ μ L, 125.80ng/ μ L, 203.75ng/ μ L, also meets the subsequent experimental requirement.
2, transgenic wheat specific gene assembly A, B, C complete sequence obtain and component analysis
(1) according to as shown in table 1 from the inside and outside source of the wheat gene-correlation information design primer that GenBank obtains:
Table 1 qualitative PCR detects wheat native gene, the required primer sequence of foreign gene
Using TaKaRa? LA?
DNA? Polymerase (Code? No.DRR002A), extracted in Step 1 Sample C genomic DNA as a template, the primer pair for PCR amplification crossing, there may be amplified by each of the components from each 5μl a 3% agarose gel electrophoresis, the results as shown in Figure 1.Use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) that electrophoretic band is reclaimed order-checking then, obtain each element sequences.
On above-mentioned working foundation, further obtain sequence between each element, method is following:
Reagents: Use a variety of TaKaRa PCR? Kit, such as TaKaRa? LA?
DNA? Polymerase (Code? No.DRR002A), TaKaRa? LA?
With? GC? Buffer (Code? No.DRR02AG) and so on.
Template: the genomic dna that extracts the sample A obtain, B, C with step 1 is a template.
Primer: each F primer and each R primer cross match in the above-mentioned table 1.
Carry out repeatedly different condition pcr amplification, the sequence between each element of increasing, the sequence results that obtains between the ubiquitin F/uidA R is correct.Be summarized as follows:
Use TaKaRaLA
DNAPolymerase(CodeNo.DRR002A); With A genomic DNA and C genomic DNA is template; Carry out pcr amplification; Respectively get 5 μ l and carry out 3% agarose gel electrophoresis, the result as shown in Figure 2.Use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) that electrophoretic band is reclaimed order-checking, the sequence results between the ubiquitin F/uidA R is correct.
(2) flanking sequence obtains
1) carrying out flanking sequence with genomic dna obtains
To obtain the ubiquitin? F / uidA? R basis of the sequence between the designed primers flanking sequences, using TaKaRa? Genome? Walking? Kit (TaKaRa? Code? No.D316) with TaKaRa? LA?
(Code? No.DRR002A ) for ubiquitin? F / uidA? R sequences flanking sequences at both ends.
2) making up the segment storehouse carries out flanking sequence and obtains
So use Clontech Genome Walker
TMUniversal Kit (Clontech Code No.638904) makes up the segment storehouse, and pcr amplification reagent uses
2 PCR Kit (Clontech Code No.639206) carry out obtaining of flanking sequence, and progressively the step moves.Finally obtain transgenic wheat high-molecular-weight glutelin subunit transgenic wheat different strain, i.e. the edge sequences of transgene component among sample A, B, the C.
Wherein, Clontech Genome Walker
TMUniversal Kit Clontech Genome Walker
TMThe basic experiment process of Universal Kit is following:
A. genomic DNA is used Pvu II, Dra I, Stu I, EcoR V enzyme to cut respectively;
B. be connected to Genome Walker Adaptor behind the purifying respectively;
Be used for the PCR reaction after the c.TE dilution;
D. design primer and carry out pcr amplification, and analyze experimental result, reclaim the purpose band;
E. the purpose band is checked order, analyze sequencing result; To new " purpose " sequence that obtains, carry out the sequence checking;
F. repeat above-mentioned steps d～e repeatedly, until the complete sequence of accomplishing required part.
(3) sequence total length and edge sequence obtains
Obtain the edge sequence through above-mentioned experimentation, and finally obtain the target gene assembly full length sequence of each sample, described gene assembly should be the gene fragment that has comprised allogenic gene and edge sequence.
1) sample A: transgenic wheat B72-8-11b
The gene assembly A that comprises specificity exogenous array among the transgenic wheat B72-8-11b comprises uidA among this gene assembly A, ubi, lacZ 1Dx5 and wheat cdna group partial sequence.The complete sequence of this gene assembly A is shown in SEQ IDNO:11.Each component analysis result shows among this gene assembly A: gene assembly A sequence total length 10384, wherein: 1～1782nd, uidA, 1766～3794th, ubi, 3797～6475th, lacZ, 6476～8073rd, 1Dx5,8213～10384th, wheat cdna group partial sequence.
In the said gene assembly A complete sequence, the determined edge of contriver sequence, i.e. sequence shown in the SEQ ID NO:14, it is positioned at the 8213rd～10384 of SEQ ID NO:11 sequence.
2) sample B: transgenic wheat B73-6-1
The gene assembly B that comprises specificity exogenous array among the transgenic wheat B73-6-1 comprises two sections wheat cdna group partial sequence end to end among this gene assembly B, and the uidA in these two sections sequences, ubi, lacZ, 1Dx5, coliDHl.The complete sequence of this gene assembly B is shown in SEQ ID NO:12.Each component analysis result shows among this gene assembly B: gene assembly B sequence total length 14690; 1～403rd, wheat portion gene group sequence; 404～2468th, uidA; 2477～4500th, ubi; 4503～7178th, lacZ; 7177～8377th, 1Dx5,9179～11784th, coliDHl, 11786～14690th, wheat portion gene group sequence.
In the said gene assembly B complete sequence, the determined edge of contriver sequence, i.e. sequence shown in SEQ ID NO:15 and the SEQID NO:16, they lay respectively at the 1st～403 and 11786～14690 of SEQ ID NO:12 sequence.
3) sample C: transgenic wheat B102-1-2
The gene assembly C that comprises specificity exogenous array among the transgenic wheat B102-1-2; Comprise two sections wheat cdna group partial sequence end to end among this gene assembly C; And the ubi in these two sections sequences; Vector pBANF-bar; Vector pUbiGUSPlus; Vector HSP, reporter vestor pUbiGUSPlus, ubiquitin and coli DH1.The complete sequence of this gene assembly C is shown in SEQ ID NO:13.Each component analysis result shows among this gene assembly C: gene assembly C sequence total length 14042; 1～3993rd, wheat portion gene group sequence; 3994～5164th, ubi; 5220～5750th, vector pBANF-bar; 5766～6044th, vector pUbiGUSPlus; 6044～8682nd, vector HSP; 8695～9671st, reporter vestor pUbiGUSPlus; 9672～10121st, ubiquitin; 10121～10213rd, coli DH1,10214～14042nd, wheat portion gene group sequence.
In the said gene assembly C complete sequence, the determined edge of contriver sequence, i.e. sequence shown in the SEQ ID NO:17, they are positioned at the 10214th～14042 of SEQ ID NO:13 sequence.
3, the foundation of PCR detection method
Obtain the special separately detection zone in SEQ ID NOS:11～13 in order to increase, according to its separately complete sequence design PCR detect primer.Detect primer screening through too much taking turns PCR, finally obtain following Auele Specific Primer such as table 2:
Table 2
| Sequence numbering | The primer title | Length nt | Base sequence (5 '-3 ') |
| ?SEQ?ID?NO：1 | B72-F1 | 28 | ATGATGGAGGGCATTACTTGGGAGTGAA |
| ?SEQ?ID?NO：2 | B72-R1 | 25 | ACCCTGATCCCCAAAGTAAACTCGC |
| ?SEQ?ID?NO：3 | B73-FF1 | 24 | AGTATATCCGGGACGTTACAACAC |
| ?SEQ?ID?NO：4 | B73-FR1 | 26 | TATCAGTGTGCATGGCTGGATATGTA |
| ?SEQ?ID?NO：5 | B73-RF1 | 26 | CACGGCGCGTTGAATCTCCTCTGTAT |
| ?SEQ?ID?NO：6 | B73-RR1 | 25 | ATGCCTTTGTTTTCCCGCCCTCCCA |
| ?SEQ?ID?NO：7 | B102-F1 | 20 | GCGAGAGCCGCTGTATGTTC |
| ?SEQ?ID?NO：8 | B102-R1 | 22 | CGAGTAAATAATGCCAGCCTGT |
| ?SEQ?ID?NO：9 | GAG56D-F | 21 | CCCAACAACAACCACCGTTCA |
| ?SEQ?ID?NO：10 | GAG56D-R | 21 | TGGCCCTGGACGAGAGTACCT |
Wherein the amplification object of GAG56D-F/GAG56D-R is a Triticum specificity native gene.
Setting up the PCR reaction system is: cumulative volume 50 μ L, dH
2O is the basis, contains: testing sample DNA 5ng/ μ L, 10 * LA PCR BufferII (Mg
2+Plus) (TaKaRa LA
Code No, DRR002A) 5ul, each 0.4mM of dNTP, primer is to (table 3) each 0.2 μ M, Taq archaeal dna polymerase (TaKaRa LA
Code No, DRR002A) 0.05U/ul.
Above-mentioned system is carried out pcr amplification according to following condition:
94 ℃, 1min, 1 circulation;
98 ℃ of 10s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations;
72 ℃, 7min, 1 circulation.
Pcr amplification product is got 10ul, carries out 1% agarose gel electrophoresis, and the result is shown in Fig. 3 and table 3:
Table 3
Visible from this result, each designs primer can amplify the target gene fragment very accurately, specifically.
Through condition optimizing, the pcr amplification condition optimizing of B102-1-2 strain transgenic wheat sample is:
94 ℃, 1min, 1 circulation;
98 ℃ of 10s, 62 ℃ of 30s, 72 ℃ of 30s, 35 circulations;
72 ℃, 7min, 1 circulation.
Based on this, reach a conclusion:
(1) method A: the PCR to transgenic wheat B72-8-11b (sample A) detects, and uses B72-F1/B72-R1 (SEQ ID NO:1/2) to carry out pcr amplification as Auele Specific Primer, and condition is:
Cumulative volume 50 μ L, dH
2O is the basis, contains: testing sample DNA 5ng/ μ L, 10 * LA PCR BufferII (Mg
2+Plus) 5ul, each 0.4mM of dNTP, each 0.2 μ M of primer B72-F1 and B72-R1, Taq archaeal dna polymerase (TaKaRa LA
) 0.05U/ul.
Above-mentioned system is carried out pcr amplification according to following condition:
94 ℃, 1min, 1 circulation;
98 ℃ of 10s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations;
72 ℃, 7min, 1 circulation.
Through 1% agarose gel electrophoresis detected result, positive like amplification, then testing sample is transgenic wheat B72-8-11b, and is negative like amplification, and then testing sample is not transgenic wheat B72-8-11b.
(2) method B: the PCR to transgenic wheat B73-6-1 (sample B) detects, and uses B73-FF1/B73-FR1 (SEQ ID NOS:3/4) or B73-RF1/B73-RR1 (SEQ ID NOS:5/6) to carry out the PCR amplification as Auele Specific Primer, and condition is:
Cumulative volume 50 μ L, dH
2O is the basis, contains: testing sample DNA 5ng/ μ L, 10 * LA PCR BufferII (Mg
2+Plus) 5ul, each 0.4mM of dNTP, each 0.2 μ M of primer B73-FF1/B73-FR1 or B73-RF1/B73-RR1, Taq archaeal dna polymerase (TaKaRa LA
) 0.05U/ul.
Above-mentioned system is carried out pcr amplification according to following condition:
94 ℃, 1min, 1 circulation;
98 ℃ of 10s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations;
72 ℃, 7min, 1 circulation.
Through 1% agarose gel electrophoresis detected result, positive like amplification, then testing sample is transgenic wheat B73-6-1, and is negative like amplification, and then testing sample is not transgenic wheat B73-6-1.
(3) method C: the PCR to transgenic wheat B102-1-2 (sample C) detects, and uses B102-F1/B102-R1 (SEQ ID NOS:7/8) to carry out pcr amplification as Auele Specific Primer, and condition is:
Cumulative volume 50 μ L, dH
2O is the basis, contains: testing sample DNA 5ng/ μ L, 10 * LA PCR BufferII (Mg
2+Plus) 5ul, each 0.4mM of dNTP, each 0.2 μ M of primer B102-F1 and B102-R1, Taq archaeal dna polymerase (TaKaRa LA
) 0.05U/ul.
Above-mentioned system is carried out pcr amplification according to following condition:
94 ℃, 1min, 1 circulation;
98 ℃ of 10s, 62 ℃ of 30s, 72 ℃ of 30s, 35 circulations;
72 ℃, 7min, 1 circulation.
Through 1% agarose gel electrophoresis detected result, positive like amplification, then testing sample is transgenic wheat B102-1-2, and is negative like amplification, and then testing sample is not transgenic wheat B102-1-2.
1, specimen preparation:
Choose 10 of wheat samples to be measured (as shown in table 4), pulverization in liquid nitrogen uses DNA Extraction Kit for GMO Detection Ver.2.0 (TaKaRa Code No.D9093) to carry out extracting genome DNA respectively.Detect the genomic dna quality, carry out quality and concentration with agarose gel electrophoresis then, confirm that the quality of sample gene group DNA and concentration all meet the subsequent experimental requirement with ultramicrospectrophotometer (Nanodrop) detection genomic dna.
2, the method A that is set up according to embodiment 1, method B and method C detect these 10 samples, and the result is as shown in table 4, and wherein "+" expression amplification is positive, and "-" expression amplification is negative.
Can find out from the detected result of table 4,
Method A can be accurately, sensitive, detect the sample that comes from transgenic wheat B72-8-11b specifically; All the other transgenic wheats and non-transgenic wheat samples are reported as non-transgenic wheat B72-8-11b sample, conform to the sample practical situation;
Method B can be accurately, sensitive, detect the sample that comes from transgenic wheat B73-6-1 specifically; All the other transgenic wheats and non-transgenic wheat samples are reported as non-transgenic wheat B73-6-1 sample, conform to the sample practical situation;
Method C can be accurately, sensitive, detect the sample that comes from transgenic wheat B102-1-2 specifically; All the other transgenic wheats and non-transgenic wheat samples are reported as non-transgenic wheat B102-1-2 sample, conform to the sample practical situation.
Table 4
Claims (4)
1. the detection method of transgenic wheat B72-8-11b, be method through PCR to detect sequence be the gene assembly of SEQ IDNO:11, comprise in this gene assembly that sequence is the edge sequence of SEQ ID NO:14.
2. the described method of claim 1, the primer that it is characterized in that described pcr amplification is to being SEQ IDNOS:1/2.
3. the described method of claim 2 is characterized in that described PCR reaction conditions is:
94 ℃, 1min, 1 circulation;
98 ℃ of 10s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations;
72 ℃, 7min, 1 circulation.
4. the described method of claim 3 is characterized in that described PCR reaction system is 50 μ L systems, contains: testing sample DNA 5ng/ μ L, 10 * LA PCR BufferII (Mg
2+Plus) 5ul, each 0.4mM of dNTP, primer is to each 0.2 μ M, Taq archaeal dna polymerase 0.05U/ul.
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| CN114369677A (en) * | 2022-01-04 | 2022-04-19 | 江汉大学 | Primer combination, kit, detection method and application for detecting wheat transgenic components and transgenic strains |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932037A (en) * | 2006-09-18 | 2007-03-21 | 栾凤侠 | Method of screening transgenic wheat |
| CA2714790A1 (en) * | 2009-09-17 | 2011-03-17 | Commonwealth Scientific And Industrial Research Organisation | Wheat plants with immunity to wsmv |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932037A (en) * | 2006-09-18 | 2007-03-21 | 栾凤侠 | Method of screening transgenic wheat |
| CA2714790A1 (en) * | 2009-09-17 | 2011-03-17 | Commonwealth Scientific And Industrial Research Organisation | Wheat plants with immunity to wsmv |
Non-Patent Citations (1)
| Title |
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| 王娜丽等: "转基因小麦外源优质亚基基因的杂交转育研究", 《武汉植物学研究》 * |
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| CN114369677A (en) * | 2022-01-04 | 2022-04-19 | 江汉大学 | Primer combination, kit, detection method and application for detecting wheat transgenic components and transgenic strains |
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