CN110273021A - The specificity of transformant detection primer of transgenic pest-resistant herbicide-resistant corn C M8101 and its application in backcross transformation - Google Patents
The specificity of transformant detection primer of transgenic pest-resistant herbicide-resistant corn C M8101 and its application in backcross transformation Download PDFInfo
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Abstract
The invention belongs to transgenic plant detection fields, and in particular to transgenic pest-resistant herbicide-resistant corn C M8101 specificity of transformant detection primer and its apply in backcross transformation.PCR method of the invention can be used as a kind of effective means for identifying CM8101 transformant, and it is used for the backcross transformation of CM8101 transformant anti insect gene, the advantages of this method is that quick, high sensitivity, specificity is good, required experiment condition is not high, therefore has very high practical value.Supervision means are provided for the further application of CM8101.
Description
Technical field
The invention belongs to transgenic plant detection fields, and in particular to transgenic pest-resistant herbicide-resistant corn C M8101 conversion
It body specific detection primer and its is applied in backcross transformation.
Background technique
Corn is important grain, feed and insutrial crop, accounts in national food security and the national economic development
There is critical role.In recent years, China's maize sown area is maintained at more than 500,000,000 mu, be distributed mainly on national more than 20 a provinces, city, from
Control area.Since cropping system, natural climate etc. change, Ostrinia furnacalis has seriously affected per unit area yield, total yield and the product of corn
Matter becomes the important pests of China's maize production.Weeds in field and crop competition water, fertilizer, luminous energy and growing space, directly affect
Crop yield and quality.In addition, as rural laborer is toward the quickening of urban migration speed, the scale of corn planting and machine
Tool chemical conversion is trend, this makes traditional artificial weeding mode become unrealistic.Insect pest and crop smothering are the weights for influencing maize production
Factor is wanted, cultivating pest-resistant herbicide-resistant corn is the key technology approach for solving the problem, can effectively prevent corn borer etc.
Production loss is retrieved in the harm of lepidoptera pest and weeds, reduces Pesticide use amount, has huge economy, environment and society
Benefit.
Bt gene C ry1Ab is widely used at present for lepidoptera pest such as corn borer, the pest-resistant base of bollworm
Because of (http://cera-gmc.org/GMCropDatabase).In terms of transgenic corns, the commercialization of Cry1Ab gene is answered
With the most extensively.Monsanto Company MON80100, MON802, MON810；BT11, BT176 of Syngenta Co., Ltd and Du Pont are first
The transformant such as cutting edge of a knife or a sword company MON809 and its derived varieties realize commercialization and promote.The commercialization for turning Cry1Ab corn is promoted
Nearly 20 years, safety was finalized, i.e., is safety by safety evaluation, the genetically modified organism for obtaining safety certificate and products thereof
's.
The Bt anti insect gene Cry1Ab-Ma for the artificial reconstructed synthesis that Institute of Crop Science, Chinese Academy of Agricultural Science proposes,
It has obtained national inventing patent authorization (ZL 201010574708.X), bar gene is to resist to remove so far using more one
Careless agent gene.Leading chain-ordering (the L- of the protein-bonded untranslated of Wheat Chloroplasts A/B is merged using 35S constitutive promoter
Cab) and rice actin1 introne is initiated through Cry1Ab-Ma gene expression after codon optimization, by wheat HSP17 albumen
End non-translational region is as terminator；Start the expression of bar gene using 2 × P35S constitutive promoter fusion TEV enhancer,
By soybean Tvsp as terminator, building includes the plant expression vector of bar gene.Corn is transferred to using agrobacterium-mediated transformation
In genome, the transgenic corn events CM8101 of a pest-resistant herbicide-resistant is obtained, is had in terms of pest-resistant and herbicide-resistant
There is good characteristic, is likely to enter commercial growth from now on.The inspection of transformation event specificity is carried out to transgenic corns CM8101
It surveys, can preferably exercise supervision management to transgenic corns CM8101.And the flanking sequence of external source Insert Fragment and according to by this
The detection method that lateral order column are established is the important indicator of management of exercising supervision.Therefore it needs to obtain transgenic corns
The flanking sequence of CM8101, and identification system is established in case the supervision and management of part as to this.
Summary of the invention
The technical problem to be solved by the present invention is to provide detection means for CM8101 specificity of transformant.
Insect-resistant transgenic corn event CM8101 of the invention is obtained as follows:
The leading chain-ordering of the protein-bonded untranslated of Wheat Chloroplasts A/B (L-Cab) is merged using 35S constitutive promoter
Cry1Ab-Ma gene (nucleotide sequence such as SEQ ID No.14 after codon optimization is initiated through with rice actin1 introne
It is shown, wherein 228~1015bp is Southern blot probe portion) expression, it is turned over by the end of wheat HSP17 albumen is non-
Area is translated as terminator；Using 2 × P35S constitutive promoter fusion TEV enhancer starting bar gene, (nucleotide sequence is such as
Shown in SEQ ID No.15, wherein 1~548bp be Southern blot probe portion) expression, by soybean Tvsp conduct
Terminator, building include the plant expression vector of bar gene.Using agrobacterium-mediated transformation, by foreign gene and other genes member
Part imports in recipient corn material HiII, and the transformation event is transferred to corn inbred line Zheng 58 by the method for backcross transformation
Genetic background.PCR is carried out to transgenic plant to identify to obtain insect-resistant transgenic corn C M8101.Hybridize through Southern blot
Experiment and the experiment of transgenic progeny gene identification identify that the gained transgenic positive plant CM8101 is a copy.It is right
Transgenic corns carry out greenhouse and field connects Ostrinia furnacalis, show that the transgenic corns CM8101 obtained has good corn
Snout moth's larva resistance.
The present invention provides transgenic pest-resistant herbicide-resistant corn C M8101 specificity of transformant detection primer, has such as SEQ
Nucleotide sequence shown in ID No.12 and SEQ ID No.13.
The present invention also provides the backcross transformation detection primer of transgenic pest-resistant herbicide-resistant corn C M8101 anti insect gene,
The primer pair has the nucleotide sequence as shown in SEQ ID No.12 and SEQ ID No.13.
The present invention also provides the backcross transformation detection method of transgenic pest-resistant herbicide-resistant corn C M8101 anti insect gene,
During backcross transformation, PCR detection is carried out to backcross population offspring, is judged according to amplification, if amplified production item
Band size is 629bp, then is the target offspring of backcross transformation.
Beneficial effects of the present invention: the present invention expands to have obtained insect-resistant transgenic corn event CM8101 by TAIL-PCR
5 ' and 3 ' end flanking sequences design a pair of of Specific PCR primers and simultaneously establish needle according to the sequence information of the two flanking sequences
To the detection method of transgenic corns CM8101 and its product.Flanking sequence and specific primer provided by the invention are to being suitable for
To transgenic corns CM8101 (including parent, Hybrids F1, offspring and backcross transformation are from generation to generation) and its product (including plant, tissue,
Seed and its product) detection.
Detailed description of the invention
Fig. 1 is target gene Cry1Ab-Ma specific probe position and restriction enzyme site signal in T-DNA sequence
Figure.
Fig. 2 is the Cry1Ab-Ma probe Southern hybridization analysis of transgenic corns CM8101 (T5).M:Marker
(λ-DNA)；I digestion transgenic corns CM8101 of swimming lane 1:Sac；I digestion negative control Zheng 58 of swimming lane 2:Sac；Swimming lane 3:Nde I
Digestion transgenic corns CM8101；I digestion negative control Zheng 58 of swimming lane 4:Nde；Swimming lane 5: positive plasmid control.
Fig. 3 is bar specific probe position and restriction enzyme site in T-DNA sequence.
Fig. 4 is transgenic corns CM8101 (T5) bar probe Southern hybridization analysis, M:Marker (λ-DNA)；
Swimming lane 1:Hind III digestion transgenic corns CM8101；Swimming lane 2:Hind III digestion negative control Zheng 58；Swimming lane 3:Sac I
Digestion transgenic corns CM8101；I digestion negative control Zheng 58 of swimming lane 4:Sac；I digestion transgenic corns of swimming lane 5:Nde
CM8101；I digestion negative control Zheng 58 of swimming lane 6:Nde；Swimming lane 7: positive plasmid control.
The amplification of Fig. 5 specificity qualitative PCR.M:DNA molecular weight marker (II DNA marker of Trans 2k Plus)；1: matter
Grain；2: water；3: negative control Zheng 58；4:MON810；5:MON89034；6:BT176；The transgenic corns of 7:T7 generation
CM8101；The transgenic corns CM8101 of 8:T8 generation；The transgenic corns CM8101 of 9:T9 generation.
Fig. 6 specificity qualitative PCR sensitivity test.M:DNA molecular weight marker (II DNA of Trans 2k Plus
marker)；The content of 1-7:CM8101DNA is respectively 50%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%；8: water；9:
Negative control Zheng 58.
The detection of Fig. 7 backcross population Cry1Ab-Ma gene.A): backcross population BC3 generation PCR amplification；B): backcross population
BC4 generation PCR amplification；C): backcross population BC5 generation PCR amplification；M:DNA molecular weight marker (II DNA of Trans 2k Plus
marker)；1:CM8101 (T9)；2: water；3: negative control Zheng 58；4:CA193GM；5:CA509GM；6:CA667GM；7: east
401GM；8:DNF34-2GM；9:PHV37GM；10: four 144GM；11:TF9GM；12:H446GM.
Specific embodiment
1. enzyme and reagent
DNA Marker, Taq archaeal dna polymerase, plastic recovery kit, E.coli competence Trans1-T1, pEASY-
T1Simple Cloning Kit is purchased from TIANGEN company；Ex Taq enzyme, LA Taq enzyme are purchased from Takara；Agarose is purchased from
invitrogen；Roche digoxin kit is purchased from Beijing Suo Laibao Science and Technology Ltd；Primer synthesis and determining nucleic acid sequence
It is completed by Mei Ji biotech firm.
2 laboratory apparatus
PCR amplification instrument: S1000TMThermal Cycler(Bio-Rad)；Nucleic acid electrophoresis apparatus: DYY-III type nucleic acid electrophoresis
Instrument (Liuyi Instruments Plant, Beijing)；DNA electrophoretic analysis system: Gel DocTMXR+ gel imaging system (Bio-Rad)；Other instrument
Device includes: centrifuge, water-bath etc..
Integration of 1 target gene of embodiment in pest-resistant herbicide-resistant transgenic corns CM8101 genome
1, Southern blot hybridization detection technique
(1) agarose electrophoresis: taking a certain amount of DNA sample to be measured, with 10~20 μ g of digestion with restriction enzyme appropriate；
Digestion finishes, in Ago-Gel electrophoresis；After electrophoresis, transferring film.
(2) trace shifts: cut glue and mark (lower-left clipped corner) in order to position gel is then placed in a container
In, with 0.25M HCl depurination 10min；By soak in suitable denaturing liquid, 30min is placed at room temperature and is allowed to be denaturalized,
It gently shakes incessantly；By filter paper, nylon membrane be cut into it is onesize with glue, nylon membrane immerse transfer buffer in balances
30min；It successively paves, there are bubbles and fold between each layer；Siphon is shifted for 24 hours.
(3) it fixed dna: removes nylon membrane and immerses 2 × SSC solution number min, wash away the gel particle being infected on film；By film
It is clipped between two dry filter papers, 80 DEG C of baking 2-4h.
(4) film: being put into clean hybrid pipe by prehybridization with long tweezers, just facing towards suitable double steam in pipe, is added
Water keeps film wet, the bubble between membrane and tube wall is rolled with glass bar；Prehybridization solution is preheated at 65 DEG C in advance, then by 10mL
Prehybridization solution is added in hybrid pipe, tightens nozzle, detects without leakage, is fixed in hybrid heater；65 DEG C of prehybridization 2h or more.
(5) label probe: the probe sequence marked required for amplifying in the plasmid with target gene is used after amplification
0.8% Ago-Gel separation, the required probe sequence marked of gel extraction；The probe sequence purifying that recycling is obtained is dense
It is reduced to 16 μ L (containing 1 μ g DNA), is added in sterile centrifuge tube；By above-mentioned solution in 100 DEG C of boiling water water-bath 10min,
Ice bath immediately after, cooling 10min, is denaturalized DNA；The efficient DNA digoxigenin labeled and detection reagent that 4 μ L are mixed well is added
Dig-High Prime in box II is mixed, of short duration centrifugation.37 DEG C of heat preservation 20h；65 DEG C of water-bath 10min terminate reaction.
(6) hybridize: taking out hybrid pipe, 7mL hybridization solution is sucked out, 20 μ L denatured probes are added, sealed after excluding bubble；It will be miscellaneous
Traffic control is put into hybridization 12h or more in hybrid heater.
(7) film is washed by following condition: 2 × SSC, 0.1%SDS 50mL room temperature 5min/2 times；1 × SSC, 0.1%SDS
42 DEG C of 50mL 15min/2 times；56 DEG C of 0.5 × SSC, 0.1%SDS 50mL 15min/2 times；The film washed immerses in 2 × SSC
2min takes out film, the moisture content of film surface is blotted with filter paper, and wrapped up with preservative film, paying attention to cannot between preservative film and nylon membrane
There is bubble, preservative film is not superimposed.
(8) colouring method: being dyed with CSPD, is put into camera bellows and is exposed 15 minutes.
2, Cry1Ab-Ma gene insertion copy number analysis
Material uses transgenic corns CM8101 (T5) and control material Zheng 58, it is determined using the method for Southern hybridization
The copy number of foreign gene.Three kinds of single restriction endonucleases are selected to carry out digestion digestion to genomic DNA respectively, selected enzyme T- again
Only one restriction enzyme site of region of DNA, and not in the binding sequence of correspondent probe (or have multiple restriction enzyme sites, but
It is not located at probe binding sequence side), therefore each insertion point will be displayed as a special band in genome, after digestion
Southern hybridization is carried out, chooses a part of target gene respectively as probe.Each Southern hybridization includes positive right
According to and negative control sample, the control plasmid through digestion, copy equivalent is added in non-transgenic wild-type corn DNA, uses
Make positive control；Non-transgenic wild type control DNA is used as negative control.
Target gene Cry1Ab-Ma specific probe Sac I, Nde I restriction restriction enzyme site as shown in Figure 1,
Probe size is 788bp, and the Cry1Ab-Ma target gene of the probe of blot containing Southern is shown in SEQ ID NO.14.With specificity
The T of probe and the transgenic corns CM8101 of two kinds of digestions5Southern hybridization, knot are carried out for corn gene group DNA and control
Fruit obtains different size of single band (Fig. 2) respectively.Theoretically Sac I digestion hybridization, which obtains stripe size, should be greater than 5.2kb,
The practical size that obtains is about 6.2kb, meets expection；Nde I digestion hybridization, which obtains stripe size, should be greater than 5.0kb, practical to obtain
Size is about 5.7kb, meets expected (table 1).As a result it proves that Cry1Ab-Ma gene has been integrated into Maize genome, is turning
In gene corn CM8101, target gene Cry1Ab-Ma expression cassette is inserted into recipient corn genome, carrier in the form singly copied
Upper destination gene expression box framework is complete.
The size of the expection of table 1 and practical hybridising band
| Swimming lane | Sample DNA | Restriction enzyme | It is expected that copy number | It is expected that stripe size (kb) | Practical stripe size (kb) |
| 1 | CM8101 | Sac I | 1 | > 5.2 | 6.2 |
| 2 | Negative control Zheng 58 | Sac I | 0 | Nothing | Nothing |
| 3 | CM8101 | Nde I | 1 | > 5.0 | 5.7 |
| 4 | Negative control Zheng 58 | Nde I | 0 | Nothing | Nothing |
| 5 | Positive plasmid control | - | 1 | 5.4 | 5.4 |
3, the Southern hybridization and analysis of bar gene
Bar gene-specific probe and Hind III, Sac I, Nde I restriction restriction enzyme site as shown in figure 3,
Probe size is 578bp, and the bar gene of the probe of blot containing Southern is shown in SEQ ID No.15.With specific probe and three kinds
The T of the transgenic corns CM8101 of digestion3To T5Southern hybridization is carried out for corn gene group DNA and control, is as a result obtained respectively
Obtain different size of single band (Fig. 4).Theoretically the hybridization of Hind III digestion, which obtains stripe size, should be greater than 2.2kb, practical
Obtaining size is about 2.6kb, meets expection；Sac I digestion hybridization, which obtains stripe size, should be greater than 5.2kb, practical to obtain size
About 6.2kb meets expection；Nde I digestion hybridization, which obtains stripe size, should be greater than 5.0kb, and the practical stripe size that obtains is about
5.7kb meets expected (table 2).As a result prove that bar gene has been integrated into Maize genome, in transgenic corns CM8101
In, bar gene expression frame is inserted into recipient corn genome in the form singly copied, and gene expression box framework is complete on carrier.
The stripe size that table 2 is expected and is actually hybridized
| Swimming lane | Sample DNA | Restriction enzyme | It is expected that copy number | It is expected that stripe size (kb) | Practical stripe size (kb) |
| 1 | CM8101 | Hind III | 1 | > 2.2 | 2.6 |
| 2 | Negative control | Hind III | 0 | Nothing | Nothing |
| 3 | CM8101 | SacI | 1 | > 5.2 | 6.2 |
| 4 | Negative control | Sac I | 0 | Nothing | Nothing |
| 5 | CM8101 | Nde I | 1 | > 5.0 | 5.7 |
| 6 | Negative control | Nde I | 0 | Nothing | Nothing |
| 7 | Positive plasmid control | - | 1 | 5.4 | 5.4 |
The analysis of 2 external source insetion sequence of embodiment
The flanking genes group sequence of Insert Fragment is obtained by the Tail-PCR method of optimization and close to 5 ' ends and 3 ' end insertions
The sequence of Maize genome.
According to the hiTAIL-PCR method of the reports such as Liu Yaoguang, the specific primer of hiTAIL-PCR according to the end of carrier 5 ' and
3' terminal sequence designs nested type specific primer, and nucleotide sequence and expanding fragment length are shown in Table 3, make in hiTAIL-PCR
Any degenerate primer AD1-n (n is 1 to 4) and AC-1, AD6 are shown in Table 4.
3 specific primer of table
| Sequence number | Title | Primer sequence (5'-3') |
| SEQ ID No.1 | LB0-1 | AGGGGTGTGGAAATAGTGATTTGAGCACGTC |
| SEQ ID No.2 | LB1-1 | ACGATGGACTCCAGTCCAACGTCGTGACTGGGAAAACCCTGGC |
| SEQ ID No.3 | LB2-1 | TTGCAGCACATCCCCCTTTCGCCAGCT |
| SEQ ID No.4 | RB0-1 | CTCAGGATTTAGCAGCATTCCGGGTAC |
| SEQ ID No.5 | RB1-2 | GTAAAGCCTGGGGTGCCTAATGAGTGAG |
Table 4 any degenerate primer AD1-n, AC1 and AD6
Each step PCR reaction system is as shown in table 5, and hiTAIL-PCR includes 3 wheel PCR reactions, and reaction condition is shown in Table 6.
Table 5 expands PCR reaction system in advance
Table 6Tail-PCR reaction condition
Amplified production is separated with 1% agarose gel electrophoresis, by comparing the electrophoretic separation map of reaction products at different levels,
Secondary or third-order reaction specific band is cut, with general DNA QIAquick Gel Extraction Kit purification and recovery, and is cloned into pEASY-
On T1Cloning Vector, selects positive colony and be sequenced.Obtained flanking sequence is compared with carrier sequence, tentatively
Required segment is analyzed whether it is, obtained flanking sequence is compared with ncbi database then, analyzes whether it is corn-based
Because of the sequence of group.By being sequenced and utilizing DNAMAN software and ncbi database to compare, the results show that 1~146bp is carrier sequence
Column, 147~922bp are maize genomic sequence；Specific as follows: underscore is Insert Fragment sequence, and no underscore is genome
Sequence, dash area are the primer location of specific qualitative PCR detection method design:
The foundation and sensitivity test of the specific qualitative PCR detection architecture of embodiment 2
Insertion and flank corn genomic DNA sequence is held to design specific PCR detection primer according to CM8101 transformant 5 ':
8101-5 ' F:CTTATGCGGATGATGAGAGC (SEQ ID No.12)；
8101-5 ' R:AGAAAACACTGGGGAAAGAAACA (SEQ ID No.13).
Different transgenic corns and non-transgenic corn genome are used to carry out PCR amplification as template.Amplification system is such as
Under: 1 μ L, 2.5mM dNTPs of corn gene group DNA 0.8 μ L, 10 × Buffer 2 μ L, Primer-F (10 μM)/Primer-R
(10 μM) each 0.5 μ L, Taq Polymerase 0.1 μ L, ddH2O 5.1μL.PCR response procedures are as follows: 94 DEG C of 5min；94℃
30s, 60 DEG C of 35s, 72 DEG C of 45s, 35 circulations；72℃5min.
The target fragment of 629bp can be amplified only in CM8101 as the result is shown, other transgenic corns (transgenosis things
Part 1, transgenic event 2, transgenic event 3), negative control Zheng 58, water do not amplify target fragment (Fig. 5), illustrate this
Detection architecture specificity is preferably.
In order to determine specific qualitative PCR detection architecture sensitivity, it is prepared for a series of transgenic corns CM8101 contents
50%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0 powder sample respectively extracts DNA.Use transgenosis
The specific qualitative PCR detection method of corn C M8101 carries out PCR amplification, parallel three times to repeat, PCR reaction system and reaction item
Part is same as above.PCR amplification is carried out by template of dilution DNA concentration 50ng/ μ L.The results show that being 50%- in CM8101DNA content
In 0.1% range, the amplified band of expected 629bp can be obtained；When CM8101DNA content is 0.05%, do not expand
Increase apparent purpose band (Fig. 6) out.Show that this method detection sensitivity is to meet transgenosis qualitative detection greater than 0.1% and want
It asks.
The detection of 3 backcross transformation group Cry1Ab-Ma gene specific qualitative PCR of embodiment
During backcross transformation, using CM8101 specificity of transformant qualitative PCR detection primer 8101-5 ' F/R, to warp
Plant after crossing glufosinate-ammonium and the screening of Bt test strips carries out the foreground detection of Cry1Ab-Ma gene, is specifically used for the single plant of backcrossing
Middle carrying Cry1Ab-Ma gene.Molecular labeling 8101-5 ' F/R 9 backcross transformation groups three generations (BC3, BC4,
BC5) and CM8101 (T9) can amplify the segment (Fig. 7) of 629bp, it was demonstrated that backcross transformation is by Cry1Ab-Ma in CM8101
The successful transformation of gene stablizes heredity into 9 receptor self-mating systems, and in each backcross generations.
SEQUENCE LISTING
<110>Chongqing Agricultural Academy of Science
<120>the specificity of transformant detection primer of transgenic pest-resistant herbicide-resistant corn C M8101 and its in backcross transformation
Application
<130> 2019
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 31
<212> DNA
<213> artificial
<220>
<223> LB0-1
<400> 1
aggggtgtgg aaatagtgat ttgagcacgt c 31
<210> 2
<211> 43
<212> DNA
<213> artificial
<220>
<223> LB1-1
<400> 2
acgatggact ccagtccaac gtcgtgactg ggaaaaccct ggc 43
<210> 3
<211> 27
<212> DNA
<213> artificial
<220>
<223> LB2-1
<400> 3
ttgcagcaca tccccctttc gccagct 27
<210> 4
<211> 27
<212> DNA
<213> artificial
<220>
<223> RB0-1
<400> 4
ctcaggattt agcagcattc cgggtac 27
<210> 5
<211> 28
<212> DNA
<213> artificial
<220>
<223> RB1-2
<400> 5
gtaaagcctg gggtgcctaa tgagtgag 28
<210> 6
<211> 33
<212> DNA
<213> artificial
<220>
<223> LAD1-1
<220>
<221> misc_feature
<222> (24)..(24)
<223>n represents g, c or a
<220>
<221> misc_feature
<222> (25)..(25)
<223>n represents g, c, t or a
<220>
<221> misc_feature
<222> (26)..(26)
<223>n represents g, c or a
<220>
<221> misc_feature
<222> (27)..(27)
<223>n represents g, c, t or a
<220>
<221> misc_feature
<222> (28)..(28)
<223>n represents g, c, t or a
<220>
<221> misc_feature
<222> (29)..(29)
<223>n represents g, c, t or a
<400> 6
acgatggact ccagagcggc cgcnnnnnng gaa 33
<210> 7
<211> 33
<212> DNA
<213> artificial
<220>
<223> LAD1-2
<220>
<221> misc_feature
<222> (24)..(24)
<223>n represents g, c or t
<220>
<221> misc_feature
<222> (25)..(25)
<223>n represents a, g, c or t
<220>
<221> misc_feature
<222> (26)..(26)
<223>n represents g, c or t
<220>
<221> misc_feature
<222> (27)..(27)
<223>n represents a, g, c or t
<220>
<221> misc_feature
<222> (28)..(28)
<223>n represents a, g, c or t
<220>
<221> misc_feature
<222> (29)..(29)
<223>n represents a, g, c or t
<400> 7
acgatggact ccagagcggc cgcnnnnnng gtt 33
<210> 8
<211> 34
<212> DNA
<213> artificial
<220>
<223> LAD1-3
<220>
<221> misc_feature
<222> (24)..(24)
<223>n represents g, c or a
<220>
<221> misc_feature
<222> (25)..(25)
<223>n represents g, c or a
<220>
<221> misc_feature
<222> (26)..(26)
<223>n represents t, g, c or a
<220>
<221> misc_feature
<222> (27)..(27)
<223>n represents g, c or a
<220>
<221> misc_feature
<222> (28)..(28)
<223>n represents t, g, c or a
<220>
<221> misc_feature
<222> (29)..(29)
<223>n represents t, g, c or a
<220>
<221> misc_feature
<222> (30)..(30)
<223>n represents t, g, c or a
<400> 8
acgatggact ccagagcggc cgcnnnnnnn ccaa 34
<210> 9
<211> 34
<212> DNA
<213> artificial
<220>
<223> LAD1-4
<220>
<221> misc_feature
<222> (24)..(24)
<223>n represents g, c or t
<220>
<221> misc_feature
<222> (25)..(25)
<223>n represents g, a or t
<220>
<221> misc_feature
<222> (26)..(26)
<223>n represents c, g, a or t
<220>
<221> misc_feature
<222> (27)..(27)
<223>n represents c, g or t
<220>
<221> misc_feature
<222> (28)..(28)
<223>n represents a, c, g or t
<220>
<221> misc_feature
<222> (29)..(29)
<223>n represents a, c, g or t
<220>
<221> misc_feature
<222> (30)..(30)
<223>n represents a, c, g or t
<400> 9
acgatggact ccagagcggc cgcnnnnnnn aggt 34
<210> 10
<211> 16
<212> DNA
<213> artificial
<220>
<223> AC1
<400> 10
acgatggact ccagag 16
<210> 11
<211> 16
<212> DNA
<213> artificial
<220>
<223> AD6
<220>
<221> misc_feature
<222> (3)..(3)
<223>n represents A or T
<220>
<221> misc_feature
<222> (5)..(5)
<223>n represents A, G, C or T
<220>
<221> misc_feature
<222> (8)..(8)
<223>n represents A or T
<220>
<221> misc_feature
<222> (10)..(10)
<223>n represents A, G, C or T
<220>
<221> misc_feature
<222> (13)..(13)
<223>n represents G or C
<400> 11
tgngnagnan canaga 16
<210> 12
<211> 20
<212> DNA
<213> artificial
<220>
<223> 8101-5'F
<400> 12
cttatgcgga tgatgagagc 20
<210> 13
<211> 23
<212> DNA
<213> artificial
<220>
<223> 8101-5'R
<400> 13
agaaaacact ggggaaagaa aca 23
<210> 14
<211> 715
<212> DNA
<213> artificial
<220>
<223>detection sequence
<400> 14
cgtcgtctat tggaccgaca gttgctatta atataatggg ccaccatagt agactgacaa 60
ataaattacc tgacaacatc gtttcacaaa aaaacaaaca caaaaaggga gtgcattttc 120
cagggcattt ttgtaataaa aaacagttaa aagggagtgc aatagaaata taggggtgtg 180
gaaatagtga tttgagcacg tcttgaagcg aattagcttg gcactggccg tcgttttaca 240
acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 300
tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 360
cagcctgaat ggcgaatgct agagcaattc gccgttaatt cagtacatta aaaacgtccg 420
caatgtgtta gaagggtcag ctatacttgg tagattttga tagagctgaa ctcgacactt 480
gcttaattgc taagactaac atgggttggc tctggcaccg ccgactagct catgttggga 540
tgaagaatct ccataagctt ctaaagggag aacacatttt aggactaaca aatgttcatt 600
ttgagaaaga taggatttgt agcgcatgcc aagcaggaaa gcaagttggc tctcatcatc 660
cgcataagaa cataatgacg accgacaggc cactggagct cctgcacatg gatct 715
Claims (3)
1. transgenic pest-resistant herbicide-resistant corn C M8101 specificity of transformant detection primer, it is characterised in that: have such as SEQ
Nucleotide sequence shown in ID No.12 and SEQ ID No.13.
2. the backcross transformation detection primer of transgenic pest-resistant herbicide-resistant corn C M8101 anti insect gene, it is characterised in that: this draws
Object is to the nucleotide sequence as shown in SEQ ID No.12 and SEQ ID No.13.
3. the backcross transformation detection method of transgenic pest-resistant herbicide-resistant corn C M8101 anti insect gene, it is characterised in that: returning
During handing over transformation, PCR detection is carried out to backcross population offspring, is judged according to amplification, if amplified product band is big
Small is 629bp, then is the target offspring of backcross transformation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094932A (en) * | 2020-08-14 | 2020-12-18 | 浙江省农业科学院 | Qualitative PCR detection method and kit for transgenic insect-resistant corn GAB-3 transformant |
| WO2022094790A1 (en) * | 2020-11-04 | 2022-05-12 | 中国农业大学 | Corn event 2a-7 and identification method therefor |
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| CN102094030A (en) * | 2010-11-30 | 2011-06-15 | 中国农业科学院作物科学研究所 | Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof |
| CN109536490A (en) * | 2018-11-09 | 2019-03-29 | 中国农业科学院作物科学研究所 | Transgenic pest-resistant herbicide-resistant corn C M8101 external source Insert Fragment flanking sequence and its application |
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| CN102094030A (en) * | 2010-11-30 | 2011-06-15 | 中国农业科学院作物科学研究所 | Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof |
| CN109536490A (en) * | 2018-11-09 | 2019-03-29 | 中国农业科学院作物科学研究所 | Transgenic pest-resistant herbicide-resistant corn C M8101 external source Insert Fragment flanking sequence and its application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112094932A (en) * | 2020-08-14 | 2020-12-18 | 浙江省农业科学院 | Qualitative PCR detection method and kit for transgenic insect-resistant corn GAB-3 transformant |
| WO2022094790A1 (en) * | 2020-11-04 | 2022-05-12 | 中国农业大学 | Corn event 2a-7 and identification method therefor |
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