CN106244674A - A kind of standard plasmid molecule for transgenic wheat detection and construction method thereof - Google Patents
A kind of standard plasmid molecule for transgenic wheat detection and construction method thereof Download PDFInfo
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- CN106244674A CN106244674A CN201610396777.3A CN201610396777A CN106244674A CN 106244674 A CN106244674 A CN 106244674A CN 201610396777 A CN201610396777 A CN 201610396777A CN 106244674 A CN106244674 A CN 106244674A
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Abstract
The present invention relates to a kind of standard plasmid molecule for transgenic wheat detection and construction method thereof, it is characterized in that utilizing the method for over-lap PCR and molecular cloning to construct containing Semen Tritici aestivi reference gene WaXY D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar, T NOS and the standard plasmid molecule of strain B73 61 specific sequence, and prove that this standard molecule can detect for the PCR of transgenic wheat as positive criteria material by confirmatory experiment, well solve the problem of positive criteria material want during transgenic wheat detects.
Description
Technical field
The present invention is technical field of molecular biology, relates to the plasmid molecule of a kind of technical field of molecular biology, specifically
Relate to a kind of standard plasmid molecule for transgenic wheat detection and construction method thereof.
Background technology
Genetically modified crops are also known as genetic modification crop or genetically modified crops.By the improvement to gene, give certain artificially
One crop implants another kind of biological gene, thus reach to improve yield, have additional nutrients, disease-resistant, drought resisting, the mesh such as pest-resistant
's.From nineteen eighty-three whole world first case transgene tobacco since the U.S. comes out, genetically modified crops more come in the effect of agriculture field
The most important.
Semen Tritici aestivi is originated as world's main food, and its cultivated area, total output and total trade all occupy first of all kinds of crop,
The improvement of its transgene genetic receives significant attention.In cereal crops, Semen Tritici aestivi belongs to the crop of genetic transformation difficulty the most, turns base
Because research is started late.(the Rapid production of multiple independent lines such as Weeks in 1993
Of fertile transgenic wheat (Triticum aestivum L) .Plant Physiol, 1993,102 (4):
The Semen Tritici aestivi that 1077-1084) utilized particle gun mediated method by channel genes such as bar, establishes the technology body of Wheat Transformation Efficiency By Particle Bombardment
System.(the Genetic transformat of wheat mediated by Agrobacterium such as Cheng in 1997
Tumefaciens, Plant Physiol, 1997,115 (3): 971-980) utilize Agrobacterium-mediated transformation Semen Tritici aestivi to obtain first
Obtained and carried the isogenic transfer-gen plant of NPTII.The same year, by (Transformation of wheat with such as Barro F
high molecular weight subunit genes results in improved functional
Properties.Nature Biotechnology, 1997,15:1295-1299) use particle gun by pIDx5, pAHC25 two
Individual plasmid proceeds to obtain transgenic wheat B73-6-1 in wheat breed by cotransformation.This transgenic wheat strain can change
The baking properties of its bread product, has herbicide-resistant gene Bar simultaneously, has anti-grass fourth phosphine characteristic.
Carry out detection GMOs and currently mainly have two class technology: a class is nucleic acid detection method, such as PCR and gene chip
Deng, another kind of for method of protein detection, such as ELISA etc., wherein PCR detection method is most widely used.Carried out by PCR method
Detection GMOs can be divided into four kinds by strategy: the detection of general original paper selective mechanisms, gene specific, structural specificity detection and product
It it is specific detection.The general original paper selective mechanisms promoter that mainly detection transgenic is common and terminator；Gene specific
The detection exogenous genetic fragment that mainly detection is inserted；Structural specificity detection mainly detection is inserted between exogenous DNA array
The nucleotide sequence of junction；Event-specific detection predominantly detects the nucleotide sequence of foreign DNA and the junction of Plant Genome.
When carrying out PCR detection, in addition to arranging the negative control of necessity and being monitored detection, it is positive right also should to arrange
According to getting rid of PCR system inhibitive factor that may be present and the correctness of checking system.In general, PCR detection should be with from sun
Property standard variety extract genomic DNA as positive control, but due to all restrictions such as GMO bio-safety, turn base
Because crop positive criteria product are difficult to obtain, it is therefore desirable to research and develop a kind of positive control material being readily available to meet transgenic inspection
The needs surveyed.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of transgenic wheat detection standard plasmid divides
Son and construction method so that it is can substitute for qualitative, quantitative for transgenic wheat of various transgenic wheat positive criteria material
PCR detects.Special according to current Semen Tritici aestivi common transgenic element Ubiquitin, NPTII, Bar, T-NOS and strain B73-6-1
Property sequence and internal reference WaXY-D1 gene order fragment, carry out artificial sequence synthesis, and the principle then utilizing over-lap PCR to react sets
Meter specific PCR primers, obtains transgenic wheat common element and the restructuring of mono-section of sequence of reference gene WaXY-D through PCR amplification
DNA fragmentation.Molecule clone technology is utilized to be cloned into by recombinant dna fragment in pMD-19T carrier, it is thus achieved that new plasmid molecule (life
Entitled pPONY-TEST01.The standard plasmid molecule that the present invention builds can replace losing transgenic wheat original positive criteria thing
Matter, for qualitative and quantitative PCR detection, thoroughly solves a difficult problem for transgenic wheat detection Plays material want.
The present invention is achieved by the following technical solutions, standard plasmid molecule of the present invention, little to substitute transgenic
The positive criteria material of wheat, for qualitative and quantitative PCR detection.This standard plasmid molecule contains Semen Tritici aestivi common transgenic unit simultaneously
Part Ubiquitin, NPTII, Bar, T-NOS and strain B73-6-1 specific sequence and internal reference WaXY-D1 gene order.
Standard plasmid molecule pPONY-TEST01 construction method of the present invention, comprises the steps:
1) GenBank data base (https: //www.ncbi.nlm.nih.gov/genbank/) is utilized to carry out biological letter
Breath credit analysis, it is thus achieved that Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar,
T-NOS and strain B73-6-1 specific sequence.
Described Semen Tritici aestivi reference gene WaXY-D1, refers to wheat grain and combines starch synthase gene, and it is in wheat-based
Because group camber is guarded, there is specificity between planting, the feature of the interior non-specific and single copy number of kind.
Described common wheat transgenic element Ubiquitin, refers to maize ubiquitin protein gene promoter, is usually used in turning
The promoter of gene monocotyledon exogenous gene.
Described common wheat transgenic element NPTII, refers to neomycin-3 '-phosphoric acid transferase gene, it is usually used in turning base
Antibiotic-screening because of plant.
Described common wheat transgenic element Bar, refers to grass fourth phosphinothricin acetyl transferase gene, is usually used in strengthening transgenic
Plant is to herbicide grass fourth phosphine resistance.
Described common wheat transgenic element T-NOS, refers to nopaline syntase terminator, is usually used in transgenic plant
The terminator of exogenous gene.
Described wheat line B73-6-1 specific sequence, refers to wheat transgenic strain B73-6-1 external source and inserts load
Body and the DNA sequence of Wheat volatiles adjacent area.
2) design PCR specific primer.
Described PCR specific primer, generally refers to the oligonucleotide chain that length is not more than 50bp, itself and Semen Tritici aestivi internal reference base
Because WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar, T-NOS and strain B73-6-1 are special
Property sequence is identical or complementary.
3) specific amplification Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin,
NPTII, Bar, T-NOS and strain B73-6-1 specific sequence.
Described specific amplification, refers to utilize the PCR primer specific amplification Semen Tritici aestivi reference gene WaXY-of design
D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar, T-NOS and strain B73-6-1 specific sequence.
The primer that the above-mentioned PCR amplification related to uses is as follows:
4) Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar, T-
NOS and the splicing of strain B73-6-1 specific sequence.
Described splicing, refers to utilize overlapping pcr by common to Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat
Transgenic element Ubiquitin, NPTII, Bar, T-NOS and the several independent DNA fragmentation of strain B73-6-1 specific sequence are even
Connect the Novel DNA fragments being fused into a restructuring.
5) the plasmid molecule clone containing new recombinant dna fragment.
Described molecular cloning, refers to restructuring DNA fragment specific obtained above is connected to plasmid vector pMD-
On 19T, it is thus achieved that standard plasmid molecule pPONY-TEST01, its plasmid map as shown in Figure 1, sequence such as SEQ ID No:1 institute
Show.This standard molecule also leaves Pst I and HindIII restriction enzyme site, facilitates the addition of follow-up transgenic element, and will
PPONY-TEST01 imports escherichia coli EH5 α can preserve use for a long time.
6) the qualitative and quantitative PCR detecting method checking of standard plasmid molecule pPONY-TEST01.
Described qualitative PCR detection method checking, refers to the transgenic unit that standard plasmid molecule pPONY-TEST01 comprises
Part and strain specificity fragment can only expand acquisition in transgenic wheat genomic DNA, and can not at other wheat lines, and
Other genetically modified crops genomes expand and obtains.
Described quantitative PCR detecting method checking, refers to that examination criteria plasmid molecule pPONY-TEST01 is being carried out quantitatively
Sensitivity during pcr analysis, to identify that this standard molecule substitutes the ability of transgenic wheat positive criteria material.
7) present invention simultaneously provides a kind of method detecting transgenic wheat, including the standard plasmid molecule with above-mentioned structure
For positive criteria material, measure and whether wheat samples to be measured exists transgenic element to be measured and strain, and transgenic strain
Content.
Described detection method, refers to the method the using quantitative fluorescent PCR transgenic element to comprising in standard plasmid
Carried out quantitative analysis with strain by the detection primer provided and drawn standard curve, testing sample is entered by the detection primer provided
Row quantitative fluorescent PCR, judges whether containing transgenic element to be measured and strain by Ct value, if B73-6-1 transgenic strain
Testing result is positive, can obtain the content of transgenic strain also by standard curve.
Primer and probe that the above-mentioned PCR detection related to uses are as follows:
The beneficial effects of the present invention is, utilize the method for over-lap PCR and molecular cloning to construct containing Semen Tritici aestivi internal reference base
Because WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar, T-NOS and strain B73-6-1 are special
The standard plasmid molecule pPONY-TEST01 of property sequence, and prove that this standard molecule can be as positive criteria thing by confirmatory experiment
Matter, for the PCR detection of transgenic wheat, well solves positive criteria material want during transgenic wheat detects
Problem.
Accompanying drawing explanation
Fig. 1 is the signal collection of illustrative plates of the standard plasmid molecule pPONY-TEST01 that invention builds.
Fig. 2 is Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar,
T-NOS and the qualitative detection of strain B73-6-1 specific sequence.
Fig. 3 is standard plasmid molecule pPONY-TEST01 detection limit and the detection of repeatability.
Detailed description of the invention
As a example by embodiment, the present invention is described in detail below: the present embodiment is premised on technical solution of the present invention
Under implement, give detailed embodiment and process, but protection scope of the present invention be not limited to following embodiment.Under
The experimental technique of unreceipted actual conditions in row embodiment, generally operates according to normal condition, can refer to Sambrook etc. and write
Molecular Cloning: A Laboratory guide (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 2012) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1: the structure of standard plasmid molecule
In GenBank search for Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin,
NPTII, Bar, T-NOS and strain B73-6-1 specific sequence information.
1) the PCR primer sequential design of standard plasmid molecule pPONY-TEST01 is built
According to obtain Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin,
NPTII, Bar, T-NOS and strain B73-6-1 specific sequence, utilize software Primer 5.0 to design qualitative PCR primer, be used for
Amplification Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar, T-NOS and product
It it is B73-6-1 specific sequence.Concrete primer sequence see table.
2) Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar, T-
NOS and the amplification of strain B73-6-1 specific sequence
According to above-mentioned primer, with containing Ubiquitin, NPTII, Bar, T-NOS sequence and the transgenic of B73-6-1 strain
Semen Tritici aestivi is template, to Semen Tritici aestivi reference gene WaXY-D1, transgenic wheat common transgenic element Ubiquitin, NPTII, Bar,
T-NOS and strain B73-6-1 specific sequence carry out PCR amplification, finally obtain the amplified fragments of each element.Amplification is obtained
Band carries out reclaiming purification, shows that the sequence obtained is consistent with purpose amplified fragments sequence by sequencing analysis.
Above-mentioned PCR reaction system is:
Described PCR reaction condition is 95 DEG C of 10min, 1 circulation；95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, 40 circulations,
72℃ 10min。
3) utilize overlapping PCR method that above-mentioned six fragments are attached
Six fragment sequences obtained with above-mentioned PCR are template, with forward primer and the B73-6-1 product of WaXY-D1 gene
It is the reverse primer primer pair as over-lap PCR of specific sequence, carries out PCR amplification,
Realize the restructuring splicing of six fragments.
Concrete PCR reaction system is two steps, and first step PCR is reacted with six fragment sequences reclaimed as template, without
Primer carries out PCR amplification, particularly as follows:
Described PCR reaction condition is 95 DEG C of 10min, 1 circulation；95 DEG C of 30s, 42 DEG C of 50s, 72 DEG C of 4min, 10 circulations,
72℃ 10min。
Second step PCR reacts with first step PCR reaction mixture as template, adds primer and carries out PCR amplification, particularly as follows:
Described PCR reaction condition is 95 DEG C of 10min, 1 circulation；95 DEG C of 30s, 56 DEG C of 30S, 72 DEG C of 2min, 35 are followed
Ring, 72 DEG C of 10min.The band obtaining amplification carries out reclaiming purification.
4) recombinant dna fragment clone enters pMD-19T carrier
The recombinant fragment that above-mentioned connection obtains is connected on plasmid vector pMD-19T by the method utilizing molecular cloning, even
Connect reaction system as follows:
Above-mentioned mixed liquor 42 DEG C 90 seconds, through heat-shock transformed bacillus coli DH 5 alpha competent cell, after carrying out blue white macula screening
Take positive bacteria and drop into performing PCR qualification, identify that correct positive colony checks order, finally give standard plasmid molecule pPONY-
TEST01。
The brief collection of illustrative plates of the standard plasmid molecule pPONY-TEST01 built is as shown in Figure 1.Figure pPONY-TEST01 table
Show the carrier building standard plasmid molecule, insert Genetic elements and be followed successively by: Semen Tritici aestivi reference gene WaXY-D1, transgenic element
Ubiquitin, NPTII, Bar, T-NOS, strain B73-6-1 specific sequence；Also have after strain B73-6-1 specific sequence
Pst I and HindIII restriction enzyme site, facilitate the addition of follow-up transgenic element.
Embodiment 2: the plasmid control molecule of structure application in actually detected
1) qualitative detection of standard plasmid molecule pPONY-TEST01
According to each element sequences of standard plasmid molecule pPONY-TEST01 built, design fluorescent PCR detecting primer and spy
Pin, specific as follows:
Standard plasmid molecule is diluted to 2x109Copies/ μ l, as template, uses said elements detection primer, enters
Row quantitative fluorescent PCR.
Described real-time fluorescence PCR reaction system is:
Described real-time fluorescence PCR reaction condition is 95 DEG C of 10min, 1 circulation；95 DEG C, 15s, 58 DEG C of 30s, 40 are followed
Ring, collects fluorescence when 58 DEG C/30s of circulation every time.
Testing result display Semen Tritici aestivi reference gene WaXY-D1, transgenic element Ubiquitin, NPTII, Bar, T-NOS,
Strain B73-6-1 specific sequence is the most effectively expanded, and sees accompanying drawing 2.
2) detection limit of standard plasmid molecule pPONY-TEST01 and repeatability measure
Quasi-plasmid molecule pPONY-TEST01 is diluted to 2x10 successively4copies/μl、2x103copies/μl、
2x102copies/μl、2x101Copies/ μ l and 2x100Copies/ μ l carries out the test of repeatability and reproducibility, Mei Genong
Degree uses strain B73-6-1 detection primer carry out 3 parallel reactions and be repeated 3 times, and result shows that standard plasmid molecule concentration is
2x100During copies/ μ l, Ct value average out to 35.80, for effectively amplification, show the inspection of standard plasmid molecule pPONY-TEST01
Rising limit, up to 2 copies, has good susceptiveness.The Ct value marks that repeat between experiment obtain different with 3 times between 3 parallel reactions
Quasi-deviation is substantially all less than 0.2, illustrates that the pPONY-TEST01 standard plasmid molecule according to building has good repeatability,
See accompanying drawing 3.
The standard plasmid molecule pPONY-TEST01 that the present invention builds is to Semen Tritici aestivi reference gene WaXY-D1, transgenic element
Ubiquitin, NPTII, Bar, T-NOS, the detection primer of strain B73-6-1 specific sequence all have available good amplification
Effect, and there is good repeatability, completely can be as the positive criteria substance migration of wheat transgenic composition detection.
Claims (3)
1. the standard plasmid molecule for transgenic wheat detection and construction method thereof, it is characterised in that Semen Tritici aestivi reference gene
WaXY-D1 fragment, transgenic element Ubiquitin, NPTII, Bar, T-NOS fragment, wheat transgenic strain B73-6-1 are special
Property sequence be sequentially connected in series, after wheat transgenic strain B73-6-1 specific sequence also have Pst I and HindIII restriction enzyme site,
The sequence of this tandem sequence is as shown in SEQ ID No:1.
A kind of standard plasmid molecule for transgenic wheat detection the most according to claim 1 and construction method thereof, its
It is characterised by:
1) the Semen Tritici aestivi reference gene WaXY-D1 described in, refers to wheat grain and combines starch synthase gene, and it is at wheat cdna
Group camber is guarded, and has specificity between planting, the feature of the interior non-specific and single copy number of kind；
2) the common wheat transgenic element Ubiquitin described in, refers to maize ubiquitin protein gene promoter, is usually used in turning base
Promoter because of monocotyledon exogenous gene；
3) the common wheat transgenic element NPTII described in, refers to neomycin-3 '-phosphoric acid transferase gene, it is usually used in transgenic
The antibiotic-screening of plant；
4) the common wheat transgenic element Bar described in, refers to grass fourth phosphinothricin acetyl transferase gene, is usually used in strengthening transgenic and plants
Thing is to herbicide grass fourth phosphine resistance；
5) the common wheat transgenic element T-NOS described in, refers to nopaline syntase terminator, is usually used in outside transgenic plant
The terminator of source gene；
6) the wheat line B73-6-1 specific sequence described in, refers to wheat transgenic strain B73-6-1 exogenous insertion vector
DNA sequence with Wheat volatiles adjacent area.
3., for standard plasmid molecule and the construction method thereof of transgenic wheat detection, it is characterized by described standard plasmid
Molecule can be applied as positive criteria material.
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Cited By (1)
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