CN105274229B - Detect the method and kit of 11 foreign gene homozygosis of transgenic corns T4/heterozygous state - Google Patents
Detect the method and kit of 11 foreign gene homozygosis of transgenic corns T4/heterozygous state Download PDFInfo
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- CN105274229B CN105274229B CN201510728614.6A CN201510728614A CN105274229B CN 105274229 B CN105274229 B CN 105274229B CN 201510728614 A CN201510728614 A CN 201510728614A CN 105274229 B CN105274229 B CN 105274229B
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention provides a kind of detection method of homozygosis/heterozygous state for the insertion of 11 foreign genes of transgenic corns T4.The present invention devises PCR detection primers., as template, PCR amplification is carried out with the primer, according to the DNA fragmentation amplified from the DNA that detected sample extracts, it may be determined that go out homozygosis/heterozygous state of foreign gene insertion.Present invention also offers the PCR detection kit of homozygosis/heterozygous state for measuring the insertion of 11 foreign genes of transgenic corns T4.
Description
Technical field：
The present invention relates to a kind of detection side of homozygosis/heterozygous state to the insertion of transgenic corns T4-1-1 foreign genes
Method, the invention further relates to the kit used in for above-mentioned detection.
Background technology
In the detection of genetically modified crops, do not only need to know in sample whether contain transgene component, which kind of turns base containing
Because of component, also need to confirm the homozygosis or heterozygous state that exogenous DNA is inserted into sometimes.The qualitative detection of this transgene component is also known as
For selective mechanisms.
In detection of GMOs as, exogenous DNA can be regarded to a gene locus.In homozygotic 2 times of body genomes
Target nucleic acid sequence is detected, i.e. the copy number of exogenous DNA insertion characteristic sequence is 2；External source in 2 times of body genomes of heterozygote
The copy number of DNA insertion characteristic sequences is 1；It is then 0 in non-transgenic material.Therefore, during detection GMOs, it is necessary to original
The hereditary capacity of target nucleic acid sequence is confirmed in material, that is, it is homozygous to confirm characteristic sequence caused by exogenous DNA insertion
Or heterozygous state.In addition, during genetically modified crops genetic breeding, generally require by transgenic line and other materials into
Row hybridization, transformation are to obtain suitable cultivar, using molecular biology method is quick, easy identification homozygote/heterozygote
Breeding material, is also beneficial to shorten breeding process.
Do not stop so far, both at home and abroad also without the detection for transgenic corns T4-1-1 foreign genes homozygosis/heterozygous state
Whether method, can only determine in transgenic corns T4-1-1 samples containing transgene component or containing which kind of transgene component.
Therefore, the homozygosis of exogenous DNA insertion or the method for heterozygous state in corn T4-1-1 samples can directly be differentiated by finding
And corresponding detection reagent is developed, it is necessary.
The content of the invention
The object of the present invention is to provide a kind of homozygosis/heterozygous state for the insertion of transgenic corns T4-1-1 foreign genes
Detection method, and develop kit used in for above-mentioned detection.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
The present invention devises a kind of homozygosis/heterozygosis shape for being used to detect the insertion of transgenic corns T4-1-1 foreign genes first
The PCR detection primers of state.According to transgenic corns T4-1-1 external source Insert Fragment flanking sequence information, in maize chromosome part
The PCR detections for designing a pair of homozygosis/heterozygous state that can accurately detect the insertion of transgenic corns T4-1-1 foreign genes are drawn
Thing, its nucleotide sequence are respectively shown in SEQ ID No.1 and SEQ ID No.2.
The present invention provides a kind of detection of homozygosis/heterozygous state for the insertion of transgenic corns T4-1-1 foreign genes
Method, its step are：
(1) DNA of rice sample to be detected is extracted；
(2) using the DNA of said extracted as template, it is with the nucleotides sequence row shown in SEQ ID No.1 and SEQ ID No.2
Amplimer, establishes PCR amplification system, and carries out PCR amplification；
If only amplifying the fragment of nucleotide sequence shown in a SEQ ID No.3, homozygous transgenic corn is accredited as
T4-1-1；
If amplifying two bar segments of respectively nucleotide sequence shown in SEQ ID No.3 and SEQ ID No.4, identification
For Heterozygous transgenic corn T4-1-1；
If only amplifying the fragment of nucleotide sequence shown in a SEQ ID No.4, it is not transgenic corns to be accredited as
T4-1-1。
Wherein, the method that DNA is extracted from corn sample to be detected, can be that DNA is extracted from vegetable material
Various common methods, such as can be CTAB methods, SDS methods or empirical tests be suitable for DNA of plants extraction kit etc. it is various
Method.
Heretofore described PCR amplification system refers to following methods foundation：The cumulative volume of reaction system is 25.0 μ L,
Wherein, 10 × LA Taq Buffer II (Mg2+Plus) buffer solution 2.5 μ L, TaKaRa LA Taq (5U/ μ l) polymerase 0.25
4.0 μ L of μ L, dNTP Mixture (2.5mM each), primer 1 μ L, 10 μm of ol/L shown in 10 μm of ol/L SEQ ID No.1
1 μ L, 25ng/ μ L DNA profilings of primer, 2 μ L shown in SEQ ID No.2, surplus is distilled water.
The PCR amplification condition is preferably：94 DEG C of denaturation 1min；(94 DEG C of 20s, 65 DEG C of 5min) 30 circulations；72℃
Extend 5min.
Present invention also offers a kind of homozygosis/heterozygous state for the insertion of transgenic corns T4-1-1 foreign genes
PCR detection kit, it is characterized in that including a pair of of PCR detection primers, the nucleotide sequence of the primer is respectively SEQ ID
Shown in No.1 and SEQ ID No.2.
Detection method can accurately detect homozygosis/heterozygosis of transgenic corns T4-1-1 foreign genes insertion
State, is transgenic corns T4-1-1 composition detections Developments of certified reference samples, transgenosis quantitatively detects, sample identification has established base
Plinth.
Brief description of the drawings
Fig. 1 PCR methods identify transgenic corns T4-1-1 homozygote agarose gel electrophoresis figures；Wherein
M：Trans 2K plus II DNA Marker；1：Blank control；2：T4-1-1 homozygotes；3：T4-1-1 acceptors
With T4-1-1 homozygote biased samples；4：Negative control.
Embodiment
The invention will now be further described with reference to specific embodiments, these embodiments are only exemplary, this area skills
Art personnel should be understood that without departing from the spirit and scope of the invention can be to the details and shape of technical solution of the present invention
Formula is modified or replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
The foundation of the PCR detection method of 1 transgenic corns T4-1-1 foreign genes homozygosis of embodiment/heterozygous state
1st, design of primers and sequence
The transformant external source integrated structure is located on No. 1 chromosome of Maize genome, wherein, primer T4-1-1-F is located at 5 '
Hold on Maize genome；Primer T4-1-1-R is on 3 ' end Maize genomes.
Primer sequence
T4-1-1-F：TTAGCAGAGTCGTGAGAGTTTAG(SEQ ID No.1)
T4-1-1-R：TAGTAAATGTGATGAAACCATGAA(SEQ ID No.2)
2nd, pcr amplification reaction system and response procedures：
Reaction system：10×LA Taq Buffer II(Mg2+Plus) 2.5 μ L, TaKaRa LA Taq (5U/ μ of buffer solution
L) 4.0 μ L, 1 μ of primer shown in 10 μm of ol/L SEQ ID No.1 of 0.25 μ L, dNTP Mixture (2.5mM each) of polymerase
L, 1 μ L, 25ng/ μ L DNA profilings of primer, the 2 μ L shown in 10 μm of ol/L SEQ ID No.2, is mended to 25 μ L with deionized water.
PCR amplification program：94 DEG C of denaturation 1min；(94 DEG C of 20s, 65 DEG C of 5min) 30 circulations；72 DEG C of extension 5min.
3rd, result judgement
It is only capable of amplifying the fragment of a 4419bp in homozygous transgenic corn T4-1-1；Heterozygous transgenic corn T4-1-1
Two bar segments are amplified at the same time, one is 4419bp, and another is 386bp；And one can only be amplified in non-transgenic corn
386bp fragments.
The application experiment of the PCR detections of 2 transgenic corns T4-1-1 foreign genes homozygosis of embodiment/heterozygous state
First, vegetable material
1st, transgenic corns T4-1-1 homozygotes；
2nd, non-transgenic rice：T4-1-1 acceptors；
3rd, T4-1-1 acceptors and transgenic corns T4-1-1 homozygote biased samples.
2nd, experimental method
(1), the extraction and detection of plant genome DNA
1. vegetable material DNA is extracted
Transgenic corns are carried out using plant genome DNA extracts kit DP305 (Tiangeng biochemical technology Co., Ltd)
T4-1-1 and its recipient corn extracting genome DNA and purifying.Extraction step is referring to kit specification.
2.DNA is detected
During using UV spectrophotometer measuring DNA concentration and purity, DNA should have notable absworption peak at OD260,
OD260/OD280 ratios should be 1.7-1.9.
(2), the foundation of PCR reaction systems and response procedures
Carried out according to the pcr amplification reaction system described in embodiment 1 and response procedures.
3rd, experimental result
Experimental result is as shown in Figure 1；Homozygote T4-1-1 is only capable of amplifying the fragment of a 4419bp；T4-1-1 acceptors and
T4-1-1 homozygote biased samples amplify two bar segments, and one is 4419bp, and another is 386bp；Non-transgenic corn is only
A 386bp fragment can be amplified.
Claims (6)
1. a kind of PCR detection primers pair for being used to detect transgenic corns T4-1-1 foreign genes homozygosis/heterozygous state, its nucleosides
Acid sequence is respectively shown in SEQ ID No.1, SEQ ID No.2.
2. a kind of PCR detection kit for the homozygosis/heterozygous state for being used to measure the insertion of transgenic corns T4-1-1 foreign genes,
It is characterized in that include the PCR detection primers pair described in claim 1.
3. a kind of method of homozygosis/heterozygous state of detection transgenic corns T4-1-1 foreign genes insertion, its step are：
(1) DNA of transgenic corns T4-1-1 samples to be detected is extracted；
(2) it is to draw with the nucleotides sequence row shown in SEQ ID No.1 and SEQ ID No.2 using the DNA of said extracted as template
Thing, establishes PCR amplification system, and carries out PCR amplification；
(3) according to the DNA fragmentation amplified, homozygosis/heterozygous state that foreign gene is inserted into is determined：
If only amplifying the fragment of nucleotide sequence shown in a SEQ ID No.3, homozygous transgenic corn T4-1- is accredited as
1；
If amplifying two bar segments of respectively nucleotide sequence shown in SEQ ID No.3 and SEQ ID No.4, it is accredited as miscellaneous
Close transgenic corns T4-1-1.
The nucleotide of sequence shown in 4.SEQ ID No.1 and SEQ ID No.2 is preparing detection transgenic corns T4-1-1 external sources
Application in the PCR detection reagents of homozygosis/heterozygous state of gene insertion.
The nucleotide of sequence shown in 5.SEQ ID No.1 and SEQ ID No.2 is in detection transgenic corns T4-1-1 foreign genes
Application in homozygosis/heterozygous state of insertion.
The nucleotide of sequence shown in 6.SEQ ID No.3 and SEQ ID No.4 is in detection transgenic corns T4-1-1 foreign genes
Application in homozygosis/heterozygous state of insertion.
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| CN110195122A (en) * | 2019-01-02 | 2019-09-03 | 四川省农业科学院生物技术核技术研究所 | It is a kind of for detecting the nucleic acid sequence and its detection method of corn plant T anti-4 |
| CN114015682B (en) * | 2021-11-16 | 2022-10-21 | 四川省农业科学院生物技术核技术研究所 | Specific probe, primer, kit and method for identifying nucleic acid sample |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102206632A (en) * | 2011-03-11 | 2011-10-05 | 山东省农业科学院植物保护研究所 | Para-gene for exogenous insertion vector of transgenic maize transformation event MON88017 and application thereof |
| CN102586442A (en) * | 2012-03-02 | 2012-07-18 | 山东省农业科学院植物保护研究所 | Specific primer and probe for assaying genetically modified corn MIR162 and application thereof |
| CN103525810A (en) * | 2013-09-18 | 2014-01-22 | 上海交通大学 | Exogenous gene insertion site flanking sequence of phytase transgenic corn and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206632A (en) * | 2011-03-11 | 2011-10-05 | 山东省农业科学院植物保护研究所 | Para-gene for exogenous insertion vector of transgenic maize transformation event MON88017 and application thereof |
| CN102586442A (en) * | 2012-03-02 | 2012-07-18 | 山东省农业科学院植物保护研究所 | Specific primer and probe for assaying genetically modified corn MIR162 and application thereof |
| CN103525810A (en) * | 2013-09-18 | 2014-01-22 | 上海交通大学 | Exogenous gene insertion site flanking sequence of phytase transgenic corn and detection method |
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