CN104032017A - Primer pair for detecting G2-aroA genetically modified herbicide-tolerant corn G1105E-823C - Google Patents
Primer pair for detecting G2-aroA genetically modified herbicide-tolerant corn G1105E-823C Download PDFInfo
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Abstract
The invention discloses a primer pair for detecting G2-aroA genetically modified herbicide-tolerant corn G1105E-823C. The primer pair for detecting G2-aroA genetically modified herbicide-tolerant corn G1105E-823C provided by the invention specifically consists of two single-stranded DNA molecules shown as a sequence 1 and a sequence 2 in a sequence table. The experiment proves that whether corn to be detected is the G2-aroA genetically modified herbicide-tolerant corn G1105E-823C can be detected by utilizing the primer pair, and the method is high in accuracy rate, high in specificity and high in sensitivity.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of primer pair for detection of turning G2-aroA gene herbicide-resistant corn G1105E-823C.
Background technology
Weeds are large evils that farm crop produce, and since control of weeds technology in modern age appears in nineteen forty-two, chemical herbicide has a great development.Glyphosate class weedicide is a kind of broad spectrum, nonselective herbicide, it is by suppressing EPSPS (5-enol pyruvoyl oxalic acid-3-phosphate synthase, an important enzyme in die aromatischen Aminosaeuren route of synthesis in plant materials) activity, blocked the biosynthesizing of plants shikimic acid approach, strongly inhibited cell fission, all has strong restraining effect to many annual and perennial weedss.Because glyphosate is easy to be decomposed by microorganism, in soil, without residual hazard, to animal toxicological harmless, since Roundup in 1976 succeeds in developing, be widely used.
But, because the gramineous crops such as corn are responsive to glyphosate, its application is restricted.Therefore, glyphosate tolerant gene is proceeded to corn, not only can expand the use range of glyphosate, and can reduce production costs, protection corn is avoided poisoning, finally reaches the object of increasing both production and income.Although glyphosate tolerant gene G2-aroA was found in 2004, and can high efficient expression in host Pseudomonas fluorescens G2 and intestinal bacteria, show characteristic (the Yichen Sun of high glyphosate tolerant, Min Lin and Yiping Wang.Novel AroA with high tolerance to Glyposate, encoded by a gene of Pseudomonas putida4G-1isolated from an extremelypolluted environment in China.Aplied and environmental microbiology.2005, 71 (8): 4771-4776), but it is plant, particularly in important food crop because expression amount is compared with low and be not utilized, therefore being badly in need of application in plant to it studies, as carry out codon optimized, to accelerate it, be applied in agriculture production.
The present inventor place team is in previous work, to after the optimization of glyphosate tolerant gene G2-aroA codon, proceed in corn, obtain transgenic corns, and through experiment confirm with codon optimized before compare, the expression amount that transgenic corns general performance after codon optimized goes out G2-aroA albumen obviously improves, and the tolerance of glyphosate is also obviously improved to (Chinese patent, application number 201210107071.2, Granted publication CN102676553B).
Summary of the invention
An object of the present invention is to provide for detection of or auxiliary detection corn to be measured whether be the primer pair that turns G2-aroA gene herbicide-resistant corn G1105E-823C.
In the present invention, described in, turning G2-aroA gene herbicide-resistant corn G1105E-823C, to be specially the deposit number that is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) be the corn strain of CGMCC No.9154.
Provided by the present invention for detection of or auxiliary detection corn to be measured be whether that primer pair two single strand dnas shown in sequence 1 and sequence 2 in sequence table that turn G2-aroA gene herbicide-resistant corn G1105E-823C form.
In the present invention, in described primer pair, two single strand dnas both can be packed separately, also can wait mole hybrid packed.
Wherein, described sequence 1 is comprised of 26 Nucleotide; Sequence 2 is comprised of 26 Nucleotide.
Described primer pair is for the preparation of detecting or whether auxiliary detection corn to be measured is that the application turning in the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C also belongs to protection scope of the present invention.
Further object of the present invention be to provide a kind of for detection of or auxiliary detection corn to be measured whether be the test kit that turns G2-aroA gene herbicide-resistant corn G1105E-823C.
Provided by the present invention for detection of or auxiliary detection corn to be measured whether be the test kit that turns G2-aroA gene herbicide-resistant corn G1105E-823C, specifically can include primer pair as above.
Described test kit also can contain the internal reference primer pair that two single strand dnas shown in sequence 4 and sequence 5 form in sequence table (for the primer pair of reference gene ADH design in corn).
In described test kit, also can contain PCR and react required conventional reagent, as archaeal dna polymerase, dNTP etc.
The preparation method of described test kit also belongs to protection scope of the present invention.
The preparation method of described test kit, for following (a1) or (a2):
(a1) comprise the steps: two single strand dnas of described primer pair to pack separately respectively;
(a2) comprise the steps: two single strand dnas of described primer pair, and two single strand dnas of described internal reference primer pair are packed separately respectively.
Described primer pair, or whether described test kit is that the application turning in G2-aroA gene herbicide-resistant corn G1105E-823C also belongs to protection scope of the present invention in detection or auxiliary detection corn to be measured.
Another object of the present invention is to provide a kind of detection or whether auxiliary detection corn to be measured is the method that turns G2-aroA gene herbicide-resistant corn G1105E-823C.
Whether detection provided by the present invention or auxiliary detection corn to be measured is the method that turns G2-aroA gene herbicide-resistant corn G1105E-823C, specifically can comprise the steps:
(1) take the genomic dna of described corn to be measured is template, adopts described primer pair to carry out pcr amplification, obtains PCR product;
(2) according to the size of described PCR product, determine as follows whether described corn to be measured is to turn G2-aroA gene herbicide-resistant corn G1105E-823C: if contain size in described PCR product, be the DNA fragmentation of 820bp, described corn to be measured be or candidate for turning G2-aroA gene herbicide-resistant corn G1105E-823C; If not containing size in described PCR product is the DNA fragmentation of 820bp, described corn to be measured not for or candidate not for turning G2-aroA gene herbicide-resistant corn G1105E-823C.
Further, the DNA fragmentation that described size is 820bp is specially DNA fragmentation shown in sequence 3 in sequence table.
In described method, the annealing temperature of carrying out pcr amplification with described primer pair specifically can be 62 ℃.
More concrete, reaction conditions that the described primer pair of take carries out pcr amplification is 94 ℃ of 5min; 94 ℃ of 40s, 62 ℃ of 30s, 72 ℃ of 40s, 35-40 circulation; 72 ℃ of 10min.
In addition, while carrying out pcr amplification with described primer pair, in described primer pair, upstream and downstream primer is etc. and mole uses (as above downstream primer consumption is 250nm).
More concrete, reaction system that the described primer pair of take carries out pcr amplification is 2 * Easy Taq PCR SuperMix10 μ l, each 0.5 μ l (consumption is 250nm) of upstream and downstream primer, template DNA 1 μ l (100ng), ddH
2o8 μ l.
An also object of the present invention is to provide the flanking sequence that turns G2-aroA gene herbicide-resistant corn G1105E-823C external source Insert Fragment.
The flanking sequence that turns G2-aroA gene herbicide-resistant corn G1105E-823C external source Insert Fragment provided by the present invention is 3 ' flanking sequence; Described 3 ' flanking sequence is specially sequence 3 in sequence table.
The present invention obtains the primer pair shown in sequence 1 and sequence 2 in sequence table according to the flanking sequence design that turns G2-aroA gene herbicide-resistant corn G1105E-823C.Experiment showed, and utilize this primer pair whether can detect corn to be measured for turning G2-aroA gene herbicide-resistant corn G1105E-823C by the method for PCR, and the method accuracy rate is high, high specificity, highly sensitive.
Accompanying drawing explanation
Fig. 1 is for detecting the specific detection result of the test kit that turns G2-aroA gene herbicide-resistant corn G1105E-823C.Wherein, M is DNA molecular amount standard D2000marker; 1,2,5,7,9,10,11,12: turn G2-aroA gene herbicide-resistant corn G1105E-823C CGMCC No.9154; 3,4,6,8,13,14,15,16: 8 of embodiment 1 acquisition other proceed to the T of mG2-aroA gene
6for transgenic corns strain (non-G1105E-823C strain); WT: transformation receptor corn variety combines 31.
Fig. 2 is for detecting the sensitivity detected result of the test kit that turns G2-aroA gene herbicide-resistant corn G1105E-823C.Wherein, M is DNA molecular amount standard D2000marker; 1-5: concentration is respectively the genomic dna of 50000,5000,500,50,5 copies; CK-: the negative control that replaces template DNA with water.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, turn acquisition and the macroscopical identification of G2-aroA gene herbicide-resistant corn G1105E-823C
One, turn the acquisition of G2-aroA gene herbicide-resistant corn G1105E-823C
In earlier stage Chinese patent (the application number 201210107071.2 of applying for according to the present inventor place team, Granted publication CN102676553B) embodiment 2 (bulletin text the 36th section to the 88th section) operates, and obtains the T that several proceed to mG2-aroA gene
6for transgenic corns strain, one of them strain is designated as to G1105E-823C.
Two, turn the macroscopical identification of G2-aroA gene herbicide-resistant corn G1105E-823C
Several that step 1 is obtained proceed to the T of mG2-aroA gene
6for transgenic corns strain, carry out glyphosate resistance.Concrete operations are as follows:
1, capable long, the 3 row districts of test design: 5M, repeat density: 60 * 35cm for 3 times.
2, test is processed: according to approximately 6 times of recommendation glyphosate concentration---and the consumption of 1200ml/ mu sprays agriculture and reaches (Roundup, containing 41% glyphosate, field recommendation dosage is 150-250ml/ mu).
3, test toeatment period: 5-6 leaf phase.After 7 days, start observation experiment result.Each strain is observed at least 20 strains.
Result shows turn G2-aroA gene herbicide-resistant corn G1105E-823C growth normal, and other strains have different glyphosate harm.
The present inventor further by G1105E-823C strain in field planting, other economical characters except glyphosate resistance are investigated, found that G1105E-823C strain is on other economical characters, as nutritive ingredient of breeding time, plant height, output, seed etc., all and transgene receptor---corn variety is combined 31 basically identical, no difference of science of statistics.
To sum up, visible G1105E-823C strain has not only shown extremely strong glyphosate resistance, and has well kept the good economical character of receptor parent.The present inventor is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center by this G1105E-823C strain on April 29th, 2014 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), the biomaterial (strain) of ginseng Ju is G1105E-823C, scientific description is for turning G2-aroA gene herbicide-resistant corn, and its deposit number is CGMCC No.9154.
Embodiment 2, detection turn the preparation of the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C
One, turn the clone of the flanking sequence of G2-aroA gene herbicide-resistant corn G1105E-823C
The method separation of employing Genome walking has obtained turning 3 ' end flanking sequence of external source insertion gene in G2-aroA gene herbicide-resistant corn G1105E-823C.Specifically with reference to Genome walking test kit (TaKaRa Code:D316) specification sheets, operate.
Described 3' end flanking sequence, its nucleotide sequence is as shown in sequence in sequence table 3.This 3' end flanking sequence is comprised of T-DNA LB sequence fragment on conversion carrier and Maize genome sequence fragment two portions.Concrete, the 1-319 position of sequence 3 is T-DNA LB sequence fragment on conversion carrier, 320-820 position is Maize genome sequence fragment.
Two, detect the preparation of the test kit that turns G2-aroA gene herbicide-resistant corn G1105E-823C
3 ' the end flanking sequence that turns G2-aroA gene herbicide-resistant corn G1105E-823C obtaining according to step 1, the specific primer pair of design screening, for the preparation of test kit.
Test kit is by following special primer to, internal reference primer pair, and the required conventional reagent of PCR forms.
1, special primer pair
Primer pair for 3' end flanking sequence (sequence 5) design:
C5:5'-GAACCTGACTTTAGTGACCTCTGAAC-3'(sequence 1 is the reverse complementary sequence of the 795-820 position of sequence 3);
P3:5'-GGGAGAGGCGGTTTGCGTATTGGCTA-3'(sequence 2 is the 1-26 position of sequence 3).
In theory, adopt this special primer to carrying out pcr amplification can to obtain size be the object band of 820bp turning the genomic dna of G2-aroA gene herbicide-resistant corn G1105E-823C, obtain DNA fragmentation shown in sequence 3.
2, internal reference primer pair
Internal reference primer pair for the reference gene ADH in corn (alcohol dehydrogenase) design:
ADH-F:5'-TCTTGCCGTAAGTGTTGAAAC-3'(sequence 4);
ADH-R:5'-TGGGACAGATGGATGAGCTAC-3'(sequence 5).
In theory, adopt the various corn varieties of this internal reference primer pair to carry out pcr amplification, all can obtain the object band that size is about 400bp.
Embodiment 3, detection turn the specific detection of the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C
For sample this: 8 that turn that G2-aroA gene herbicide-resistant corn G1105E-823C CGMCC No.9154, embodiment 1 obtain other proceed to the T of mG2-aroA gene
6for transgenic corns strain, and transformation receptor corn variety combines 31.
The Auele Specific Primer obtaining with embodiment 2 to and internal reference primer pair respectively each is originally detected for sample, with the specificity of verifying that special primer is right.Each sample adopts identical detection method, wraps specific as follows:
From each, for sample, extract genomic dna this respectively, as template, adopt Auele Specific Primer that embodiment 2 obtains to and internal reference primer pair carry out respectively pcr amplification.The reaction system that two primer pairs adopt is consistent with response procedures.
Reaction system (20 μ l): 2 * Easy Taq PCR SuperMix10 μ l, each 0.5 μ l (consumption is 250nm) of upstream and downstream primer, template DNA 1 μ l (100ng), ddH
2o8 μ l.Wherein, 2 * Easy Taq PCR SuperMix is Beijing Quanshijin Biotechnology Co., Ltd's product, and its catalog number is AS111-13.
Response procedures: 94 ℃ of 5min; 94 ℃ of 40s, 62 ℃ of 30s, 72 ℃ of 40s, 35 circulations; 72 ℃ of 10min.
After reaction finishes, PCR product is carried out to 1% agarose gel electrophoresis.
Experiment arranges the negative control that replaces template DNA with water simultaneously.
Result as shown in Figure 1, all test samples adopt internal reference primer pair all to amplify the big or small object fragment that is about the reference gene ADH of 400bp, and adopt Auele Specific Primer to carrying out pcr amplification, only turning G2-aroA gene herbicide-resistant corn G1105E-823C CGMCC No.9154 amplification, to have obtained size be the object band of 820bp, and other 8 T that proceed to mG2-aroA gene
6for transgenic corns strain, and transformation receptor corn variety comprehensive 31 does not all obtain object band.The size that the present inventor further obtains amplification is sample presentation order-checking after the object band glue of 820bp reclaims, and result shows that the object band of 820bp is really as shown in sequence in sequence table 3.Above result shows that special primer that embodiment 2 obtains is to having stronger specificity.
The sensitivity that embodiment 4, detection turn the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C detects
Extraction turns the genomic dna of G2-aroA gene herbicide-resistant corn G1105E-823C CGMCC No.9154, then carry out 10 times of doubling dilutions, the copy number that obtains genomic dna in every μ l sample is respectively 25000,2500,250,25,2.5 series samples, and the template detecting as sensitivity is used.
Using the genome DNA sample of as above serial copy number respectively as template, adopt Auele Specific Primer that embodiment 2 obtains to carrying out respectively pcr amplification.
Reaction system (20 μ l): 2 * Easy Taq PCR SuperMix10 μ l, each 0.5 μ l (consumption is 250nm) of upstream and downstream primer, template DNA 2 μ l, ddH
2o7 μ l.Wherein, 2 * Easy Taq PCR SuperMix is Beijing Quanshijin Biotechnology Co., Ltd's product, and its catalog number is AS111-13.
Response procedures: 94 ℃ of 5min; 94 ℃ of 40s, 62 ℃ of 30s, 72 ℃ of 40s, 40 circulations; 72 ℃ of 10min.
Experiment arranges the negative control that replaces template DNA with water simultaneously.
As shown in Figure 2, with special primer, to carrying out pcr amplification, it is the object band of 820bp that the genome DNA sample of 25 copy numbers can amplify size to result.The size that the present inventor further obtains amplification is sample presentation order-checking after the object band glue of 820bp reclaims, and result shows that the object band of 820bp is really as shown in sequence in sequence table 3.Above result shows that special primer that embodiment 2 obtains is to having stronger sensitivity.
Claims (10)
- For detection of or auxiliary detection corn to be measured whether be the primer pair that turns G2-aroA gene herbicide-resistant corn G1105E-823C, in sequence table, two single strand dnas shown in sequence 1 and sequence 2 form.
- 2. primer pair claimed in claim 1 is for the preparation of detecting or whether auxiliary detection corn to be measured is the application turning in the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C.
- For detection of or auxiliary detection corn to be measured whether be the test kit that turns G2-aroA gene herbicide-resistant corn G1105E-823C, include primer pair claimed in claim 1.
- 4. test kit according to claim 3, is characterized in that: described test kit also contains the internal reference primer pair that two single strand dnas shown in sequence 4 and sequence 5 form in sequence table.
- 5. the preparation method of test kit described in claim 3 or 4, for following (a1) or (a2):(a1) comprise the steps: two single strand dnas of described primer pair to pack separately respectively;(a2) comprise the steps: two single strand dnas of described primer pair, and two single strand dnas of described internal reference primer pair are packed separately respectively.
- 6. primer pair described in claim 1, or described in claim 3 or 4, test kit is detecting or whether auxiliary detection corn to be measured is the application turning in G2-aroA gene herbicide-resistant corn G1105E-823C.
- 7. whether detection or auxiliary detection corn to be measured are the method that turns G2-aroA gene herbicide-resistant corn G1105E-823C, comprise the steps:(1) take the genomic dna of described corn to be measured is template, adopts primer pair described in claim 1 to carry out pcr amplification, obtains PCR product;(2) according to the size of described PCR product, determine as follows whether described corn to be measured is to turn G2-aroA gene herbicide-resistant corn G1105E-823C: if contain size in described PCR product, be the DNA fragmentation of 820bp, described corn to be measured be or candidate for turning G2-aroA gene herbicide-resistant corn G1105E-823C; If not containing size in described PCR product is the DNA fragmentation of 820bp, described corn to be measured not for or candidate not for turning G2-aroA gene herbicide-resistant corn G1105E-823C.
- 8. method according to claim 7, is characterized in that: the DNA fragmentation that described size is 820bp is DNA fragmentation shown in sequence 3 in sequence table.
- 9. according to the method described in claim 7 or 8, it is characterized in that: the annealing temperature that the primer pair described in claim 1 of take carries out pcr amplification is 62 ℃.
- 10. turning the flanking sequence of G2-aroA gene herbicide-resistant corn G1105E-823C external source Insert Fragment, is that 3 ' flanking sequence forms, and it is characterized in that: described 3 ' flanking sequence is sequence 3 in sequence table.
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