CN104032017B - For detecting the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C - Google Patents
For detecting the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C.Provided by the present invention for detecting the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C, be specifically made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.Experiment proves, utilizes 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.
Description
Technical field
The invention belongs to biological technical field, relating to a kind of for detecting the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C.
Background technology
Weeds are large evils of production estimation, and since control of weeds technology in modern age appears in nineteen forty-two, chemical herbicide has a great development.Glyphosate-class herbicides is a kind of broad spectrum, nonselective herbicide, it is by suppressing EPSPS (5-enolpyruvyl acyl oxalic acid-3-phosphate synthase, an important enzyme in plant materials in die aromatischen Aminosaeuren route of synthesis) activity, block 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 the microorganisms, without residual hazard in soil, to animal toxicological harmless, since Roundup in 1976 succeeds in developing, be widely used.
But, because the gramineous crops such as corn are to glyphosate sensitive, make it apply and be restricted.Therefore, glyphosate-tolerant gene is proceeded to corn, not only can expand the use range of glyphosate, and can reduce production cost, protection corn, from poisoning, finally reaches the object of increasing both production and income.Although glyphosate-tolerant gene G2-aroA was found in 2004, and can high expression in host Pseudomonas fluorescens G2 and intestinal bacteria, show the characteristic (YichenSun of high glyphosate tolerant, MinLinandYipingWang.NovelAroAwithhightolerancetoGlyposat e, encodedbyageneofPseudomonasputida4G-1isolatedfromanextre melypollutedenvironmentinChina.Apliedandenvironmentalmic robiology.2005, 71 (8): 4771-4776), but it is plant, be not utilized because expression amount is lower in particularly important food crop, therefore be badly in need of studying its application in plant, as codon optimized in carried out, be applied in agriculture production to accelerate it.
The present inventor place team is in previous work, to proceed in corn after the optimization of glyphosate-tolerant gene G2-aroA codon, obtain transgenic corns, and confirm compared with before codon optimized through experiment, the expression amount that transgenic corns general performance after codon optimized goes out G2-aroA albumen significantly improves, and the tolerance of glyphosate is also significantly improved (Chinese patent, application number 201210107071.2, Authorization Notice No. CN102676553B).
Summary of the invention
An object of the present invention is to provide for detect or whether auxiliary detection corn to be measured is the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C.
In the present invention, turn G2-aroA gene herbicide-resistant corn G1105E-823C described in and be specially the corn strain that the deposit number being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) is CGMCCNo.9154.
Provided by the present invention for detect or auxiliary detection corn to be measured whether for the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.
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 made up of 26 Nucleotide; Sequence 2 is made up of 26 Nucleotide.
Whether described primer pair is being that the application turned in the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C also belongs to protection scope of the present invention for the preparation of detection or auxiliary detection corn to be measured.
Further object of the present invention is to provide a kind of for detect or whether auxiliary detection corn to be measured is the test kit turning G2-aroA gene herbicide-resistant corn G1105E-823C.
Provided by the present invention for detect or whether auxiliary detection corn to be measured is the test kit turning G2-aroA gene herbicide-resistant corn G1105E-823C, specifically can include primer pair as above.
Described test kit also can containing the internal reference primer pair be made up of two single strand dnas shown in sequence in sequence table 4 and sequence 5 (primer pair for reference gene ADH in corn designs).
Conventional reagent needed for also can reacting containing PCR in described test kit, 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, is following (a1) or (a2):
(a1) comprise the steps: two of described primer pair single strand dnas individually to pack;
(a2) comprise the steps: two of described primer pair single strand dnas, and two single strand dnas of described internal reference primer pair are individually packed.
Described primer pair, or whether described test kit is that the application turned 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 turning G2-aroA gene herbicide-resistant corn G1105E-823C.
Whether detection provided by the present invention or auxiliary detection corn to be measured are the method turning G2-aroA gene herbicide-resistant corn G1105E-823C, specifically can comprise the steps:
(1) with the genomic dna of described corn to be measured for template, adopt described primer pair to carry out pcr amplification, obtain PCR primer;
(2) according to the size of described PCR primer, determine whether described corn to be measured is turn G2-aroA gene herbicide-resistant corn G1105E-823C as follows: if in described PCR primer containing size be the DNA fragmentation of 820bp, then described corn to be measured for or candidate for turning G2-aroA gene herbicide-resistant corn G1105E-823C; If in described PCR primer not containing size be the DNA fragmentation of 820bp, then 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 the process, the annealing temperature of carrying out pcr amplification with described primer pair specifically can be 62 DEG C.
More concrete, the reaction conditions carrying out pcr amplification with described primer pair is: 94 DEG C of 5min; 94 DEG C of 40s, 62 DEG C of 30s, 72 DEG C 40s, 35-40 circulation; 72 DEG C of 10min.
In addition, when carrying out pcr amplification with described primer pair, in described primer pair, upstream and downstream primer to be etc. and mole to use (as above downstream primer consumption is 250nm).
More concrete, the reaction system of carrying out pcr amplification with described primer pair is: 2 × EasyTaqPCRSuperMix10 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 turning G2-aroA gene herbicide-resistant corn G1105E-823C external source Insert Fragment.
The flanking sequence turning 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 by the primer pair shown in sequence in sequence table 1 and sequence 2 according to the flanking sequence design turning G2-aroA gene herbicide-resistant corn G1105E-823C.Experiment proves, utilizes 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 detects the specific detection result turning the test kit of 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-823CCGMCCNo.9154; 3,4,6,8,13,14,15,16: embodiment 1 obtain 8 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 detects the sensitivity technique result turning the test kit of 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 replacing template DNA with water.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, the acquisition turning G2-aroA gene herbicide-resistant corn G1105E-823C and macroscopical identification
One, the acquisition of G2-aroA gene herbicide-resistant corn G1105E-823C is turned
According to the Chinese patent (application number 201210107071.2 of the present inventor place team application in early stage, Authorization Notice No. CN102676553B) embodiment 2 (the 36th section to the 88th section of bulletin text) operate, obtain the T that several proceed to mG2-aroA gene
6for transgenic corns strain, one of them strain is designated as G1105E-823C.
Two, the macroscopical identification of G2-aroA gene herbicide-resistant corn G1105E-823C is turned
Several acquisition step one proceed to the T of mG2-aroA gene
6glyphosate resistance is carried out for transgenic corns strain.Concrete operations are as follows:
1, capable long, the 3 row districts of test design: 5M, repeat for 3 times, density: 60 × 35cm.
2, test process: according to about 6 times of recommendation glyphosate concentration---the consumption of 1200ml/ mu sprays agriculture and reaches (Roundup, containing the glyphosate of 41%, field recommendation dosage is 150-250ml/ mu).
3, toeatment period is tested: the 5-6 leaf phase.Observation experiment result is started after 7 days.Each strain observes at least 20 strains.
Result shows, and turn G2-aroA gene herbicide-resistant corn G1105E-823C and grow normal, and other strains has different glyphosate to endanger.
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 the nutritive ingredient etc. of breeding time, plant height, output, seed, all and transgene receptor---corn variety is combined 31 basically identical, no difference of science of statistics.
To sum up, visible G1105E-823C strain not only shows extremely strong glyphosate resistance, and well maintains the excellent economical character of receptor parent.This G1105E-823C strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 29th, 2014 and (is called for short CGMCC by the present inventor, 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 CGMCCNo.9154.
Embodiment 2, detection turn the preparation of the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C
One, the clone of the flanking sequence of G2-aroA gene herbicide-resistant corn G1105E-823C is turned
Adopt the method for Genomewalking to be separated and obtain and turn 3 ' the end flanking sequence that external source in G2-aroA gene herbicide-resistant corn G1105E-823C inserts gene.Concrete reference Genomewalking test kit (TaKaRaCode:D316) specification sheets operates.
Described 3' holds flanking sequence, and its nucleotide sequence is as shown in sequence in sequence table 3.This 3' holds flanking sequence to be made up of T-DNALB sequence fragment on conversion carrier and maize genomic sequence fragment two portions.Concrete, the 1-319 position of sequence 3 is T-DNALB sequence fragment on conversion carrier, and 320-820 position is maize genomic sequence fragment.
Two, the preparation turning the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C is detected
According to 3 ' the end flanking sequence turning G2-aroA gene herbicide-resistant corn G1105E-823C that step one obtains, the specific primer pair of design screening, for the preparation of test kit.
Test kit by following special primer to, internal reference primer pair, and conventional reagent composition needed for PCR.
1, special primer pair
Primer pair for 3' holds flanking sequence (sequence 5) to design:
C5:5'-GAACCTGACTTTAGTGACCTCTGAAC-3'(sequence 1, the reverse complementary sequence for the 795-820 position of sequence 3);
P3:5'-GGGAGAGGCGGTTTGCGTATTGGCTA-3'(sequence 2, the 1-26 position for sequence 3).
In theory, adopting this special primer can obtain to carrying out pcr amplification to the genomic dna turning G2-aroA gene herbicide-resistant corn G1105E-823C the object band that size is 820bp, namely obtaining DNA fragmentation shown in sequence 3.
2, internal reference primer pair
Internal reference primer pair for the reference gene ADH (alcoholdehydrogenase) in corn designs:
ADH-F:5'-TCTTGCCGTAAGTGTTGAAAC-3'(sequence 4);
ADH-R:5'-TGGGACAGATGGATGAGCTAC-3'(sequence 5).
In theory, adopt the various corn variety 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: turn G2-aroA gene herbicide-resistant corn G1105E-823CCGMCCNo.9154, embodiment 1 obtains 8 other proceed to the T of mG2-aroA gene
6for transgenic corns strain, and transformation receptor corn variety combines 31.
The Auele Specific Primer obtained by embodiment 2 to and internal reference primer pair originally detect, to verify the specificity that special primer is right for sample each respectively.Each sample adopts identical detection method, wraps specific as follows:
Extract genomic dna for sample this from each respectively, as template, the Auele Specific Primer adopting embodiment 2 to obtain to and internal reference primer pair carry out pcr amplification respectively.The reaction system that two primer pairs adopt is consistent with response procedures.
Reaction system (20 μ l): 2 × EasyTaqPCRSuperMix10 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 × EasyTaqPCRSuperMix is Beijing Quanshijin Biotechnology Co., Ltd's product, and its catalog number is AS111-13.
Response procedures: 94 DEG C of 5min; 94 DEG C of 40s, 62 DEG C of 30s, 72 DEG C of 40s, 35 circulations; 72 DEG C of 10min.
After reaction terminates, PCR primer is carried out 1% agarose gel electrophoresis.
Experiment arranges the negative control replacing template DNA with water simultaneously.
Result as shown in Figure 1, all test sample employing internal reference primer pairs have all amplified the object fragment that size is about the reference gene ADH of 400bp, and adopt Auele Specific Primer to carrying out pcr amplification, only have and turn G2-aroA gene herbicide-resistant corn G1105E-823CCGMCCNo.9154 amplification and obtain the object band that size is 820bp, and the T that other 8 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 obtains increasing further 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.
Embodiment 4, detection turn the sensitivity technique of the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C
Extract the genomic dna turning G2-aroA gene herbicide-resistant corn G1105E-823CCGMCCNo.9154, then 10 times of doubling dilutions are carried out, the copy number obtaining genomic dna in every μ l sample is respectively the series of samples of 25000,2500,250,25,2.5, and the template as sensitivity technique uses.
Respectively using the genome DNA sample of as above serial copy number as template, adopt Auele Specific Primer that embodiment 2 obtains to carrying out pcr amplification respectively.
Reaction system (20 μ l): 2 × EasyTaqPCRSuperMix10 μ l, upstream and downstream primer each 0.5 μ l (consumption is 250nm), template DNA 2 μ l, ddH
2o7 μ l.Wherein, 2 × EasyTaqPCRSuperMix is Beijing Quanshijin Biotechnology Co., Ltd's product, and its catalog number is AS111-13.
Response procedures: 94 DEG C of 5min; 94 DEG C of 40s, 62 DEG C of 30s, 72 DEG C of 40s, 40 circulations; 72 DEG C of 10min.
Experiment arranges the negative control replacing template DNA with water simultaneously.
As shown in Figure 2, with special primer to carrying out pcr amplification, the genome DNA sample of 25 copy numbers can amplify the object band that size is 820bp to result.The size that the present inventor obtains increasing further 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 (11)
1., for detect or whether auxiliary detection corn to be measured is the primer pair turning G2-aroA gene herbicide-resistant corn G1105E-823C, be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.
2. whether primer pair according to claim 1 is being turn the application in the test kit of G2-aroA gene herbicide-resistant corn G1105E-823C for the preparation of detection or auxiliary detection corn to be measured.
3., for detect or whether auxiliary detection corn to be measured is the test kit turning G2-aroA gene herbicide-resistant corn G1105E-823C, include primer pair according to claim 1.
4. test kit according to claim 3, is characterized in that: described test kit is also containing the internal reference primer pair be made up of two single strand dnas shown in sequence in sequence table 4 and sequence 5.
5. the preparation method of test kit described in claim 3, comprises the steps: two of described primer pair single strand dnas individually to pack.
6. the preparation method of test kit described in claim 4, comprise the steps: two of described primer pair single strand dnas, and two single strand dnas of described internal reference primer pair is individually packed.
7. whether test kit described in primer pair described in claim 1 or claim 3 or 4 is turn the application in G2-aroA gene herbicide-resistant corn G1105E-823C in detection or auxiliary detection corn to be measured.
8. whether detection or auxiliary detection corn to be measured are the method turning G2-aroA gene herbicide-resistant corn G1105E-823C, comprise the steps:
(1) with the genomic dna of described corn to be measured for template, adopt primer pair described in claim 1 to carry out pcr amplification, obtain PCR primer;
(2) according to the size of described PCR primer, determine whether described corn to be measured is turn G2-aroA gene herbicide-resistant corn G1105E-823C as follows: if in described PCR primer containing size be the DNA fragmentation of 820bp, then described corn to be measured for or candidate for turning G2-aroA gene herbicide-resistant corn G1105E-823C; If in described PCR primer not containing size be the DNA fragmentation of 820bp, then described corn to be measured not for or candidate not for turning G2-aroA gene herbicide-resistant corn G1105E-823C.
9. method according to claim 8, is characterized in that: the DNA fragmentation that described size is 820bp is DNA fragmentation shown in sequence in sequence table 3.
10. method according to claim 8 or claim 9, is characterized in that: the annealing temperature of carrying out pcr amplification with primer pair described in claim 1 is 62 DEG C.
11. turn the flanking DNA of G2-aroA gene herbicide-resistant corn G1105E-823C external source Insert Fragment, be 3 ' flanking DNA, it is characterized in that: the nucleotide sequence of described 3 ' flanking DNA is as shown in sequence in sequence table 3.
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