CN105483252B - The PCR detection primers and detection method of transgenic line containing gus reporter gene - Google Patents
The PCR detection primers and detection method of transgenic line containing gus reporter gene Download PDFInfo
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Abstract
The present invention provides a pair of PCR primers for being used to detect the transgenic line containing gus reporter gene, is the pair of primers according to gus gene sequences Design, its nucleotide sequence such as SEQ ID NO:Shown in 1 and 2, this pair of primer can not only detect gus reporter gene fragment (the SEQ ID NO in transgenic line by traditional qualitative PCR method:3), while distinctive one section of sequence (SEQ ID NO in rice genome can be amplified:4), thus can simultaneously as detection oryza sativa genomic dna reference gene mark.PCR detection primers and detection method that the present invention is provided, be can be not only used for detecting the transgenic sample containing gus reporter gene, and the quality for determining whether templet gene group DNA derives from Oryza and its genomic DNA is can be used for again.This method is simple, quick, and cost is low, and experimental result is visual and clear.
Description
Technical field
The present invention relates to molecular Biological Detection technology, specifically, it is related to the transgenic line containing gus reporter gene
PCR detection primers and detection method.
Background technology
Paddy rice is one of most important cereal crops in the world, is also the staple food crop of China.As population increases
With the development of society, the arable land produced available for agricultural product is fewer and fewer, has been difficult to meet need by traditional breeding technique
Ask, want to improve rice yield, quality and solve world food shortage problem, only by the improvement of crop gene engineering technology and
Select high-yield crop kind.Transgenic plant genetic engineering technology, be according to breeding objective will artificial separation and modified it is excellent
Target gene, is imported in recipient plant genome by genetic transformation, foreign gene is expressed in recipient plant, so that soon
Speed, the kind for directly obtaining heritable merit.In current agricultural production, compared with conventional breeding, transgenic technology
With incomparable advantage：1st, available genetic resources is widened, New idioplasm resource is created；2nd, plant trait can be determined
To fixed point variation and selection；3rd, to cultivate high yield, high-quality, high anti-improved seeds new way is provided (Li Wenfeng, Ji Jing (2010) is carried
The progress Scientia Agricultura Sinicas 09 of high Study of Marker Genes in Transgenic Plants security policies:1761-70).
Although transgenic technology has the advantages that many, but in China, most of transgenic product still can not direct business
Industry is, it is necessary to receive strict supervision.Therefore it is accomplished by detecting product.With the development of transgenic technology, external source base
The detection method of cause also increases therewith, is roughly divided into the nucleic acid level detection based on genetically modified plants and protein level detects two classes
Means (Querci M, Van den Bulcke M (2010) New approaches in GMO detection.Anal
Bioanal Chem 396(6):1991-2002).The test experience process of wherein protein level is complicated, time-consuming longer, is unfavorable for
The detection of transgene component rapidly and efficiently.Although the transgenosis test strips developed in recent years are more quick protein level detection sides
Method, but be only capable of detecting a limited number of albumen such as Bt albumen, EPSPS albumen etc..Detection based on nucleic acid level has due to it
Easy to detect, system is ripe, wide detectable scope and the features such as high flux, thus using it is relatively broad (Elenis D,
Kalogianni D(2008)Advances in molecular techniques for the detection and
quantification of genetically modified organisms.Anal Bioanal Chem 392(3):
347-54).Detection method based on nucleic acid level can be divided into following several again by concrete application：1st, traditional qualitative PCR detection
Technology；2nd, fluorescence quantitative PCR detection technique；3rd, multiplex PCR detection technique；4th, nucleic acid hybridization technique is detected, including Southern
Hybridization and dot hybridization；5th, gene chip detecting technique etc..Wherein traditional qualitative PCR detection is used when being converted according to genetically modified crops
(NOS is terminated for promoter such as cauliflower mosaic virus promoter (CAMV 35S promoters), nopaline syntase terminator
Son), selected marker Neomycin Transferase gene (NPTII), the cross-linked structure of exogenous promoter or terminator and crop gene group
Gene and reporter gene such as beta-glucuronidase (β-glucuronidase, GUS) gene etc. designs specific primer, uses
The method qualitative detection transgenic sample of amplification in vitro.This method is easy to operate, time-consuming short, can set up for certain crop
Standardize detection architecture (Xu CJ, Yang L (2005) Development of a rapid, reliable and simple
multiplex PCR assay for early detection of transgenic plant materials.Acta
Physiol Plant 27(3):283-8), it is most easy, ripe foreign gene detection method in genetically modified crops.
The genomic DNA template for extracting detected sample is the leading step detected into performing PCR, therefore, is obtained more high-quality
The genomic DNA of amount is the accuracy and reliability important prerequisite for ensureing PCR detections.It is generally necessary to be inhaled to genomic DNA
Light value detects that its concentration detects its integrality and palliating degradation degree with purity or agarose gel electrophoresis.Reference gene is marked
Internal reference, can indicate the accuracy of experimental result as detection DNA concentration and the object of reference of purity.If the one of design
It can detect that transfer-gen plant can be marked as the reference gene for determining templet gene group source and quality again to primer, can be significantly
Saving time and running cost, improve the detection efficiency of transgenic sample.
The content of the invention
It is described it is an object of the invention to provide the PCR primer that a pair are used for transgenic line of the detection containing gus reporter gene
Primer can not only detect external source reporter gene, and whether can contain reference gene according in electrophoresis result, further determine that
Whether templet gene group DNA derives from the quality of Oryza and its genomic DNA.
It is a further object of the present invention to provide the transgenic line containing gus reporter gene set up based on above-mentioned primer
PCR detection method.
In order to realize the object of the invention, the present invention provides the PCR detection primers of the transgenic line containing gus reporter gene,
It includes：
Forward primer：5’-TCAGTGGCAGTGAAGGG-3’(SEQ ID NO:1)
Reverse primer：5’-GAGCGTCGCAGAACATTA-3’(SEQ ID NO:2)
This pair of primer is, using gus gene sequence as according to designing, to utilize SEQ ID NO:1 and SEQ ID NO:2 enter performing PCR
Amplification, except part gus gene sequence can be obtained (amplified production size is 538bp)；It can also be used to expand transgenic paddy rice
Whether the reference gene (amplified production size is 1491bp) in sample, be the foundation of paddy rice, while can refer to as detection sample
Show the quality of genomic DNA.And this pair of primer does not have varietY specificity, (such as 11, day is spent in rice varieties in long-grained nonglutinous rice, japonica rice
This fine, winter a beautiful gem, 93-11, MH63 etc.) in be applicable.The transgenic line containing gus reporter gene is to carry GUS report bases
Because of (SEQ ID NO:3) carrier, such as plant binary expression vector DX2182；Reported by what genetic transformation was obtained containing this GUS
Callus, seedling, seed of gene etc..
The present invention also provides the kit for being used for containing above-mentioned primer detecting the transgenic line containing gus reporter gene.
The kit also includes dNTPs, Taq archaeal dna polymerase, Mg2+, PCR reaction buffers, standard positive template etc.
At least one of.
The present invention also provides application of the kit in transgenic line of the detection containing gus reporter gene.Described turn
Genetic material includes the genetically modified crops such as transgenic paddy rice, corn, wheat, soybean, cotton.
The present invention also provides the PCR detection method of the transgenic line containing gus reporter gene, comprises the following steps：
1) DNA in testing sample is extracted；
2) using step 1) in extract DNA as template, utilize above-mentioned primer progress pcr amplification reaction；
3) PCR primer is analyzed.
PCR reaction systems are calculated as with 20 μ l：10 × PCR reaction buffers (Mg2+Plus)2μl；DNTPs (each 2.5mM) 1 μ
l；10 μM of μ l of forward primer 1；10 μM of μ l of reverse primer 0.5；The μ l of template DNA 2；The μ l of 1U/ μ l Taq archaeal dna polymerases 0.5；
ddH2O complements to 20 μ l.
PCR response procedures are：95 DEG C 5 minutes；94 DEG C 30-45 seconds, 55 DEG C 30-45 seconds, 72 DEG C 1.5 minutes, common 30-35
Circulation；72 DEG C 10 minutes.
Whether gus reporter gene and genetic background can be carried with quick detection and identification transgenic sample using the above method
Whether it is Oryza.
Step 3) in row agarose gel electrophoresis are entered to PCR primer, if only amplifying a 538bp (SEQ ID NO:
3) band of size, then show that the sample is the transgenic line containing gus reporter gene, but genetic background not paddy rice；If
Two band of 538bp and 1491bp sizes are amplified simultaneously, then it is the transgenosis water containing gus reporter gene to show testing sample
Rice；If only amplifying a 1491bp (SEQ ID NO:4) band of size, then it is non-transgenic paddy rice to show the sample.
Reference gene, its nucleotide sequence such as SEQ ID NO are detected the present invention further provides a kind of rice genome:4
It is shown.
Specific PCR primers for expanding above-mentioned reference gene, it includes：
Forward primer：5 '-TCAGTGGCAGTGAAGGG-3 ' and
Reverse primer：5’-GAGCGTCGCAGAACATTA-3’.
The present invention is that can detect whether external source gus reporter gene in transgenic line inserts plant using regular-PCR method
Genome, while the primer provided using the present invention can directly be entered with amplifying rice genome characteristic sequences to rice genome
Using this sequence as reference gene during line flag, i.e. PCR primer electrophoresis, whether genomic templates DNA can be intuitively told
For paddy rice and judge templet DNA quality, the uncertainty of template DNA quality when eliminating regular-PCR amplification.Therefore, originally
The PCR detection primers and detection method provided is provided, can be not only used for detecting the transgenic sample containing gus reporter gene, can use again
In it is determined that whether templet gene group DNA derives from the quality of Oryza and its genomic DNA.This method is simple, quick, and cost is low,
Experimental result is visual and clear.
Brief description of the drawings
Fig. 1 is the PCR testing results of the transfer-gen plant containing gus reporter gene in the embodiment of the present invention 2；Wherein, left side
First swimming lane-：H2O (negative control)；The swimming lane of left side second-：ZH11；The swimming lane of left side the 3rd+：DX2182 carriers；1-12：
DX2182 different transgenic lines；Ref：Reference gene amplified fragments；GUS：Gus gene amplified fragments.
Fig. 2 is the PCR testing results of reference gene in different rice varieties in the embodiment of the present invention 3；Wherein, M：
2000marker；1：H2O；2：ZH11；3：Nipponbare；4：Dongjin；5：93-11；6：MH63；7：H2O；8：ZH11；9：
DX2182。
Fig. 3 is primer sensitivity technique result in the embodiment of the present invention 4；Wherein, M：2000marker；1-8：Transgenosis water
Rice material DNA concentration extension rate is followed successively by 100-10-7。
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning:A laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.
The acquisition of transgenic line of the embodiment 1 containing gus reporter gene
Exemplified by 11 (ZH11) are spent in rice varieties, gus reporter gene sequence (SEQ ID NO will be carried:3) carrier,
Such as DX2182 plant binary expression vectors, in the callus by Agrobacterium-mediated genetic transformation method rice transformation, utilize
Hygromycin resistance is screened, and positive transgenic plant is obtained through breaking up, taking root.
The detection of transgenic line of the embodiment 2 containing gus reporter gene
1st, the design and synthesis of primer
With gus gene sequence (SEQ ID NO:3) it is foundation, is designed and drawn inside a pair of gus genes using software Primer5
Thing GUS (DX) F1：5‘-TCAGTGGCAGTGAAGGG-3’；GUS(DX)R1：5‘-GAGCGTCGCAGAACATTA-3’.Primer expands
Increase fragment length 538bp.
2nd, DNA is extracted
Randomly select 11 plants of ZH11 paddy rice resistant plants and choose non-transgenic rice varieties ZH11, Nipponbare,
Dongjin, 93-11, MH63, extract genomic DNA respectively.DNA extraction steps are as follows：The rice leaf of about 2cm length is taken to be placed in
In 2ml centrifuge tubes；Add 800 μ 1.5 × CTAB of l in mortar, grinding blade is to being homogenized and refund in centrifuge tube；65 DEG C of water-baths
20-30min, it is reverse per 5min to mix 1 time；Add isometric chloroform/isoamyl alcohol (24:1), turn upside down mixing, continue
10min；10000rpm centrifuges 10min；400 μ l supernatants are drawn to new centrifuge tube, 95% of 2 times of volumes through ice precooling is added
Ethanol, -20 DEG C of ice put 20min；12000rpm centrifuges 15min；Supernatant is abandoned, the ethanol of 500 μ l 75% is added, rinsing is overturned,
12000rpm centrifuges 5min；Supernatant is abandoned, super-clean bench drying is placed in or dries naturally, plus 100 μ l ddH2O dissolving DNAs.
3rd, the detection of transgenic paddy rice
Using GUS (DX) F1/R1 as primer, respectively with ZH11 paddy rice resistant plant genomic DNAs (detected sample), conversion
DNA (positive control) used, non-transgenic ZH11 genomic DNAs (negative control) are template, enter performing PCR amplification.PCR is anti-
Answer program and system as follows：
PCR reaction systems are calculated as with 20 μ l：10 × PCR reaction buffers (Mg2+Plus)2μl；DNTPs (each 2.5mM) 1 μ
l；10 μM of μ l of forward primer 1；10 μM of μ l of reverse primer 0.5；The μ l of template DNA 2；The μ l of 1U/ μ l Taq archaeal dna polymerases 0.5；
ddH2O complements to 20 μ l.
PCR response procedures are：95 DEG C 5 minutes；94 DEG C 30-45 seconds, 55 DEG C 30-45 seconds, 72 DEG C 1.5 minutes, common 30-35
Circulation；72 DEG C 10 minutes.
As a result PCR primer is shown in Fig. 1 through 1.2% agarose gel electrophoresis.Electrophoresis result shows to contain in positive control
538bp bands, meet gus gene and are expected amplification length；And do not only have 538bp gus gene amplified band in transgenic paddy rice,
Equally there is the band in an also size about 1500bp band, negative control, thus it is speculated that the band is peculiar in rice genome
Sequence.
The rice genome of embodiment 3 detects the acquisition of reference gene
1st, the checking of molecule labelled series
In order to further verify whether the about 1500bp bands are rice genome characteristic sequences, japonica rice and Xian are chosen respectively
Rice Representative Cultivars (ZH11, Nipponbare, Dongjin, 93-11, MH63), using its genomic DNA as template, GUS (DX) F1/R1 is
Primer, enters the band also containing size about 1500bp after performing PCR amplification, PCR primer electrophoresis, as shown in Fig. 2 further determining that this
Sequence is characteristic sequences in rice genome.Therefore, the sequence can as gus gene augmentation detection rice genome internal reference base
Cause, indicates the quality of rice genome.
2nd, clone and analyze reference gene sequence
In order to further determine that the about 1500bp segment characterizations, above-mentioned about 1500bp fragments are tapped rubber and reclaimed, take 2 μ l to carry out
PCR electrophoresis detections purpose fragment reclaims quality, and remaining purpose fragment is connected to- carrier T (Promega), connection
System is 10 μ l, wherein carrier T 100ng, purpose fragment 200ng, the μ of 10 × coupled reaction buffer solution, 1 μ l, T4DNA ligase 0.5
l；22 DEG C of connections convert bacillus coli DH 5 alpha after 3 hours, are coated with the LB flat boards of the resistance of benzyl containing ammonia, are screened through blue hickie, 12 hours
The white monoclonal bacterial plaque of picking carries out bacterium solution PCR detections afterwards；PCR programs and system be the same as Example 2.Purpose fragment size will be met
Monoclonal bacterium solution send Hua Da gene sequencing.Sequencing shows the long 1491bp of the sequence, is entered with the sequence in Genbank databases
Row BLAST, comparison result is found, in existing gene pool, and the sequence exists only in Genus Oryza, homology 90% with
On, it was demonstrated that the sequence is rice genome characteristic sequences, its sequence such as SEQ ID NO really:Shown in 4.The sequence is located at No. 3 dyes
Colour solid, close to centriolar region, covers the two ends (AAS01940.1 and AAS01917.1) of the encoding proteins of two predictions.Its
Middle AAS01940.1 may encode an aspartic protease, and AAS01917.1 may encode a transposase, both at
Albumen is predicted, its code sequence dependent of dead military hero new gene needs further to verify the function of the gene.
The sensitivity technique experiment of the primer of embodiment 4
One plant of paddy rice ZH11 transfer-gen plant is randomly selected, DNA is extracted, DNA is detected with NanoDrop 2000 (Thermo)
Concentration is 403.6 μ g/ml.DNA sample is subjected to 10 times of gradient dilutions, 10 are followed successively by0-10-7Concentration gradient, in -20 DEG C of preservations
It is standby.
The DNA sample using above-mentioned carry out concentration gradient dilution enters performing PCR reaction, PCR reaction systems and journey as template respectively
Sequence be the same as Example 2.
Take 5 μ l PCR primers, through 1.2% agarose gel electrophoresis, in testing result if there is 538bp bands and
1491bp bands then prove that detected sample is ZH11 transfer-gen plants, illustrate that sample concentration is too low if without band.
The electrophoresis detection result of PCR reactions is shown in that Fig. 3, swimming lane 1-8 are ZH11 transfer-gen plant samples, when concentration dilution is arrived
10-5Times when band it is unobvious, be diluted to 10-6Times when do not expand to band.Therefore, the sensitivity of the primer is up to 4.036ng/
Ml, you can detect about 2pg paddy DNA, detection sensitivity is higher.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
1. the PCR detection method of the transgenic paddy rice containing gus reporter gene, it is characterised in that comprise the following steps：
1) DNA in testing sample is extracted；
2) using step 1) in extract DNA as template, utilize primer progress pcr amplification reaction；
The primer includes：Forward primer：5 '-TCAGTGGCAGTGAAGGG-3 ' and
Reverse primer：5’-GAGCGTCGCAGAACATTA-3；’
PCR reaction systems are calculated as with 20 μ l：Containing Mg2+The μ l of 10 × PCR reaction buffers 2；Each μ l of 2.5mM dNTPs 1；10μM
The μ l of forward primer 1；10 μM of μ l of reverse primer 0.5；The μ l of template DNA 2；The μ l of 1U/ μ l Taq archaeal dna polymerases 0.5；ddH2O is complemented to
20μl；
PCR response procedures are：95 DEG C 5 minutes；94 DEG C 30-45 seconds, 55 DEG C 30-45 seconds, 72 DEG C 1.5 minutes, common 30-35 is followed
Ring；72 DEG C 10 minutes；
3) PCR primer is analyzed；
Step 3) in row agarose gel electrophoresis are entered to PCR primer, if only amplifying the band of a 538bp size, table
The bright sample is the transgenic line containing gus reporter gene, but genetic background not paddy rice；If amplify simultaneously 538bp and
Two band of 1491bp sizes, then it is the transgenic paddy rice containing gus reporter gene to show testing sample；If only amplifying one
The band of bar 1491bp sizes, then it is non-transgenic paddy rice to show the sample.
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