CN107460193A - A kind of right side flap sequence of genetically engineered soybean WH8013 transformation event foreign insertion vectors - Google Patents
A kind of right side flap sequence of genetically engineered soybean WH8013 transformation event foreign insertion vectors Download PDFInfo
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- CN107460193A CN107460193A CN201710830540.6A CN201710830540A CN107460193A CN 107460193 A CN107460193 A CN 107460193A CN 201710830540 A CN201710830540 A CN 201710830540A CN 107460193 A CN107460193 A CN 107460193A
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Abstract
The present invention relates to a kind of right side flap sequence of genetically engineered soybean WH8013 transformation event foreign insertion vectors, application, primer pair, PCR kit and the primer pair of the right side flap sequence and the application method of PCR kit.Wherein, the nucleotide sequence of right side flap sequence of the present invention such as SEQ ID NO：Shown in 4, it can specifically indicate the genetically engineered soybean that pCHAL1 T DNA fragmentations are inserted at the 21177109th nucleotides of No. 8 chromosome.Present invention sequencing first discloses the right side flap sequence of genetically engineered soybean WH8013 transformation event foreign insertion vectors, establishes genetically engineered soybean WH8013 specificity qualitative PCR detection methods, this method is sensitive, accurately, simply, reliably, has wide market prospects.
Description
Technical field
The present invention relates to genetically engineered soybean field, in particular to a kind of genetically engineered soybean WH8013 transformation events outside
The right side flap sequence of source insertion vector.
Background technology
Soybean is oil crops main in the world.The cultivated area of global genetically modified crops in 2015 is up to 1.797 hundred million
Hectare, wherein soybean acreage are 0.916 hundred million hectares, account for the 51% of the gross area[1].China is not only agricultural production big country, more
It is agricultural consumption big country.Since nineteen ninety-six, China turns into soybean net importer.As domestic demand is constantly increasing, turn
Transgenic soybean import volume is also increasing year by year.Only in 2015, Chinese genetically engineered soybean import volume is up to 81,690,000 tons, increases on year-on-year basis
Long 14.4%[2]。
It is the management that exercised supervision to genetically engineered soybean to carry out scientific research and evaluation to genetically engineered soybean bio-safety problem,
Its important technical basis to develop in a healthy way is ensured, and detection GMOs technology is one of key technology of bio-safety evaluation[3],
Authentication for transfer-gen plant is significant, and helps to exist to the expression mechanism and foreign gene of foreign gene
Caused influence is studied in acceptor gene group, so as to which the biological safety helped for transfer-gen plant makes assessment[4]。
HAL1 genes are genes related to salt tolerant in saccharomyces cerevisiae, and the overexpression of the gene can improve yeast pair
NaCl stress, and knock out and adjust the gene, then substantially reduce the salt tolerance of yeast.At present, there is the plant of some HAL1 transgenosis
Thing, but not yet research obtains HAL1 genetically engineered soybeans, more lacks the method that effectively can be detected to HAL1 genetically engineered soybeans.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of right side of genetically engineered soybean WH8013 transformation event foreign insertion vectors
Flanking sequence, the right side flap sequence can specifically indicate to insert at the 21177109th nucleotides on No. 8 chromosome
The genetically engineered soybean of pCHAL1 T-DNA fragments.
The second object of the present invention is in the application in a kind of right side flap sequence of offer in genetically engineered soybean is detected, institute
Whether state to apply can be come from the 21177109th on No. 8 chromosome by the right side flap sequence with sample described in accurate judgement
The genetically engineered soybean of pCHAL1 T-DNA fragments is inserted at the nucleotides of position.
The third object of the present invention is to provide the PCR primer pair according to the right side flap sequences Design, the primer pair
By PCR reactions can quickly and accurately detect on No. 8 chromosome of the sample at the 21177109th nucleotides whether
PCHAL1 T-DNA fragments are inserted, there is the advantages of easy to detect, specific good and accuracy rate is high.
The fourth object of the present invention is to provide a kind of PCR detection kit, and the kit is by above-mentioned primer pair and its
He integrates auxiliary reagent, has the advantages of easy to use.
The fifth object of the present invention is to provide the primer pair or the application method of kit, can be with by methods described
Quickly and accurately detect the T-DNA pieces for whether inserting pCHAL1 on No. 8 chromosome of sample at the 21177109th nucleotides
Section.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of right side flap sequence of genetically engineered soybean WH8013 transformation event foreign insertion vectors, its nucleotide sequence is such as
SEQ ID NO：Shown in 4.
The present invention is established on the genetically engineered soybean WH8013 bases that Jilin Academy of Agricultural Science biology is provided.Turn base
Because soybean WH8013 is to be inserted the T-DNA of the plasmid pCHAL1 with HAL1 genes by agriculture bacillus mediated soybean cotyledon node method
Enter to the genetically engineered soybean obtained on the genome of soybean WILLIAMS-DARLING Ton 82, its good salt tolerance, economical character is good, has good
Application prospect and Breeding value.But before making the present invention, still Nobody Knows, and T-DNA is specific on genetically engineered soybean WH8013
Insertion point, it can not more establish the method that can effectively identify the genetically engineered soybean.
The present invention well-designed 3 ' SP1,3 ' SP2 and 3 ' SP3 sequences, with reference to the Genome Walking of TakaRa companies
Kit kits, the right side for obtaining genetically engineered soybean WH8013 transformation time exogenous insertion vectors is compared by TAIL-PCR and sequencing
Flanking sequence.
The nucleotide sequence of right side flap sequence of the present invention such as SEQ ID NO：Shown in 4, it includes T-DNA on pCHAL1
HAL1 gene orders, the soybean genomic sequence on the right side of 3 ' UTR sequences and the T-DNA.As can be seen here, the right side flap
Sequence had both included the T-DNA sequences of insertion vector, included the soybean genomic sequence on the right side of its insertion point again, only the 8th
The genetically engineered soybean for inserting pCHAL1T-DNA at number chromosome the 21177109th just has above-mentioned right side flap sequence, pCHAL1
And Non-transgenic soybean or other genetically engineered soybeans do not possess above-mentioned right side flap sequence.
Therefore, right side flap sequence of the present invention can be indicated specifically at No. 8 chromosome the 21177109th
Insert pCHAL1T-DNA genetically engineered soybean, especially genetically engineered soybean WH8013 or the genetically engineered soybean formed by its transformation.
Based on the sequence, it can specifically detect whether testing sample is derived from above-mentioned genetically engineered soybean, in the detection of genetically engineered soybean
It is and significant and be widely applied prospect in terms of the identification of germ plasm resource.
The invention further relates to application of the above-mentioned right side flap sequence in genetically engineered soybean is detected, the of the genetically engineered soybean
PCHAL1 T-DNA fragments are inserted at the 21177109th nucleotides of No. 8 chromosome；Preferably, the genetically engineered soybean is
WH8013 or the genetically engineered soybean by its transformation.
Application of the present invention is based on above-mentioned right side flap sequence, can pass through molecular biology method, such as PCR, molecule
Hybridization, specific detection is carried out to preceding aim genetically engineered soybean, there is simple to operate, high accuracy for examination.
In some specific embodiments, it is used to detect the genetically engineered soybean according to above-mentioned right side flap sequences Design
PCR primer pair or hybridization probe.
The invention further relates to the PCR primer pair of above-mentioned right side flap sequences Design, the nucleotide sequence of the primer pair is distinguished
Such as SEQ ID NO：6 and SEQ ID NO：Shown in 7.
Primer pair of the present invention is separately designed in the soybean gene group on the right side of T-DNA and the T-DNA insertion points,
It could only obtain 500bp or so product by the primer pair amplifies using intended transgenic soybean as template, and with
PCHAL1, Non-transgenic soybean or other kinds genetically engineered soybean are that template can not then expand acquisition target product.Therefore, originally
The PCR primer is invented to insert at the 21177109th nucleotides of No. 8 chromosome to can be used in specifically detecting
The genetically engineered soybean of pCHAL1 T-DNA fragments, especially WH8013 or the genetically engineered soybean by its transformation.It is in addition, of the invention
The primer pair also has the advantages that specificity is good, amplification efficiency is high.Therefore, primer pair of the present invention is to improving transgenosis material
The determination rates of material, the management of transgenic product and detection, and the identification of germ plasm resource have great importance.
The invention further relates to a kind of PCR detection kit, the kit includes above-mentioned primer pair, and other reagents.
Mentioned reagent box of the present invention integrates above-mentioned primer pair and other auxiliary reagents that PCR is used, and having makes
The advantages of with facilitating.
In some specific embodiments, other described reagents include archaeal dna polymerase, dNTPs, PCR reaction buffer,
One or more in positive reference substance and negative controls.
In some specific embodiments, the archaeal dna polymerase be selected from Taq, Bst, Vent, Phi29, Pfu, Tru,
Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase,
One or more in Klenow fragments.
Preferably, the archaeal dna polymerase is Taq archaeal dna polymerases.
It is highly preferred that the Taq archaeal dna polymerases are thermal starting Taq archaeal dna polymerases.
In some specific embodiments, other described reagents include the premixed liquid of PCR reactions, and the premixed liquid is
The mixture of archaeal dna polymerase, dNTPs and PCR reaction buffers.
The invention further relates to above-mentioned primer pair or the application method of mentioned reagent box, methods described to include：
(1) using the DNA of soybean sample to be measured as template, the primer pair in above-mentioned primer pair or mentioned reagent box and its are used
His reagent enters performing PCR amplification；
(2) judged according to pcr amplification product on No. 8 chromosome of the soybean to be measured at 21177109th nucleotides
Whether pCHAL1 T-DNA fragment is inserted.
The above-mentioned application method of the present invention passes through on No. 8 chromosome that simple PCR method is detectable sample the
The HAL1 genes whether inserted at 21177109 nucleotides, there is good excellent of easy to detect, specific good and accuracy
Point.
In some specific embodiments, the annealing temperature of the PCR reactions is 49~51 DEG C, and period is 28~32
It is secondary；Preferably, the annealing temperature is 50 DEG C, and the period is 30 times.
Compared with prior art, beneficial effects of the present invention are：
(1) the present invention relates to genetically engineered soybean WH8013, the 21177109th nucleosides on No. 8 chromosome of the soybean
T-DNA sequences on acid place insertion pCHAL1, so as to obtain HAL1 genes, the genetically engineered soybean can Salt And Alkali Tolerance, and table
Reveal good economical character, have broad application prospects.The present invention obtains genetically engineered soybean WH8013 transformation events first
Exogenous insertion vector right side flap sequence, design corresponding PCR primer and establish PCR method for detecting specificity, can examine exactly
Survey turn whether testing sample inserts pCHAL1 T-DNA sequences at the 21177109th nucleotides on No. 8 chromosome
Transgenic soybean, this method is sensitive, accurately, simply, reliably, has wide market prospects, the identification to improving transgenic line
Management and the detection and the identification of germ plasm resource of efficiency, transgenic product have great importance.
(2) the above-mentioned primer pair of the present invention finally determines through well-designed and repeated screening side, separately designs in T-DNA and T-
In the soybean gene group of DNA flanks, only it is inserted at the 21177109th nucleotides of No. 8 chromosome and is turned with T-DNA
Transgenic soybean is that template could expand to obtain purpose fragment, and other templates can not expand acquisition purpose fragment, can easily sentence
Whether disconnected detection object is T intended transgenic soybean.In addition, the above-mentioned primer pair of the present invention also have specificity and accuracy it is good,
The advantages of detection efficiency is high.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The required accompanying drawing used is briefly described in embodiment or description of the prior art, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 be pCHAL1 plasmids T-DNA collection of illustrative plates, wherein, the T-DNA include left margin sequence (LB), Bar genes,
HAL1 genes and right margin (RB)；
Fig. 2 is right margin Tail-PCR electrophoresis results, wherein, M1：λ-Hind III digest, 1-3：AP1 1st 2nd
3rd；4-6：AP2 1st 2nd 3rd；7-9：AP3 1st 2nd 3rd；10-12：AP4 1st 2nd 3rd；13-15：Face
According to 1st 2nd 3rd；M2：DL 2000；
Fig. 3 is right margin TAIL-PCR sequencing result, wherein, dash area is the sequence of known HAL1 genes, is added
Frame portion is divided into 3 ' UTR sequences, and italicized item is soybean genomic sequence, and right margin (RB) lacks；
Fig. 4 is soybean gene group Chr08:21177109..21177593 sequence；
Fig. 5 is genetically engineered soybean WH8013 right margin primer location figures, wherein 3 ' F designs, on T-DNA, 3 ' R designs exist
In soybean gene group on the right side of insertion point；
The electrophoresis result figure that the specific PCR that Fig. 6 is genetically engineered soybean WH8013 detects, wherein, M：Marker, 8013 are
Genetically engineered soybean WH8013 PCR reaction products.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products obtained can be bought by city.
Embodiment 1 obtains the right side flap sequence of genetically engineered soybean WH8013 insertion vectors
First, experiment material
Genetically engineered soybean WH8013 (is provided) by soybean seminar of Jilin Academy of Agricultural Science biology institute：With soybean William
This 82 is acceptor material, using plasmid pCHAL1 as conversion carrier, (that is, is turned by agriculture bacillus mediated soybean cotyledon node method for transformation
Transgenic soybean WH8013 transformation events) obtain, the collection of illustrative plates of the plasmid pCHAL1 is referring to Figure of description Fig. 1；The transgenosis
Soybean WH8013 shows Salt And Alkali Tolerance and good economical character, and the insertion point for finally determining its T-DNA through embodiment 1 is
At the nucleotides of chromosome the 21177109th of soybean No. 8.
2nd, the acquisition of right side flap sequence
1st, DNA extraction
(1) sample collection：The genetically engineered soybean WH8013 of field planting blade is taken mid-June, is protected at -80 DEG C
Deposit.
(2) DNA extraction：Extract the genomic DNA of the soybean leaves using CTAB methods, specific detection method referring to
Bibliography 5.
(3) detection of DNA concentration and purity：Use the concentration of Nano Drop detecting steps (2) described DNA sample and pure
Degree.
2nd, the acquisition of right side flap sequence
The present embodiment is obtained in genetically engineered soybean WH8013 using TaKaRa companies Genome Walking Kit kits
The flanking sequence of right margin at external source T-DNA integration sites.
(1) 3 right margin special primers (table 1) of HAL1 sequences Designs known to.
The right margin special primer of table 1
(2) using 1 μ l (500ng) transgenic soybean gene group DNA as template, with 3 ' SP1,3 ' SP2,3 ' SP3 and
AP1, AP2 and AP3, AP4 in TaKaRa Genome Walking Kit (Code No.6108) kit, and ck primers,
Carried out 5 times by TaKaRaGenome Walking Kit (Code No.6108) kit specification, 3 wheel TAIL-PCR are anti-every time
Should.
(3) electrophoresis is carried out to PCR primer.Fig. 2 shows primer AP1, AP2, AP3 the 3rd wheel PCR reactions (3,6,9 swimming lane)
Obtain providing positive control clearly specific band, 3 ' AP4-3rd of same primers amplified production band than kit
(12 swimming lanes, size are about 2.4kp) gel extraction is sequenced, that is, obtains insertion vector pCHAL1 right side flap sequence, specifically
Sequence (SEQ ID NO:4) as shown in figure 3, wherein dash area is known HAL1 gene orders, frame portion is added to be divided into 3 ' UTR sequences
Row, lack right border sequence (that is, RB sequences), italicized item is soybean genomic sequence.
3rd, the determination of external source T-DNA integration sites in soybean gene group
In soybase.org websites (https://www.soybase.org/gb2/gbrowse/gmax2.0/) comparison turn
Right flanks and soybean gene group at external source T-DNA integration sites in transgenic soybean WH8013, find the right flanks big
Beans genome C hr08:21177109..21177593(SEQ ID NO：5) it is continued presence on (Fig. 4), that is, obtains T-DNA
Integration site in soybean gene group：Soybean gene group Chr08:21177109 (that is, on No. 8 chromosome of soybean
At 21177109 nucleotides).
The genetically engineered soybean WH8013 of embodiment 2 specific PCR detection
1st, the design of PCR primer
The genetically engineered soybean WH8013 obtained according to embodiment 1 right margin flanking sequence and T-DNA sequences Design energy
The primer pair of enough specific detection T-DNA insertion events.
One primer of the primer pair is on T-DNA, and another primer is in soybean gene group：Such as 3 ' F sense primers
On T-DNA, 3 ' R anti-sense primers are on the right (Fig. 5) on boundary's genome.The F of primer pair 3 ' (SEQ ID NO：And 3R ' (SEQ ID 6)
NO：7) sequence is as shown in table 2.
The genetically engineered soybean WH8013 of table 2 specific PCR detection primer pair
2nd, genetically engineered soybean WH8013 specific PCR detection
Using genetically engineered soybean WH8013 genomic DNA as template, detected using above-mentioned PCR primer to entering performing PCR.Its
In, specific reaction condition is as follows：
1st step：94℃ 2min
2nd step：94℃ 30s
3rd step：50℃ 30s
4th step：72℃ 1min
5th step：72℃ 5min
6th step：16℃ 1h
2nd~4 step circulates 30 times.
3rd, amplified production is detected using 1% Ago-Gel, can not be expanded to band in plasmid and negative-type, only
Transgenic positive material obtains the fragment (Fig. 6) of expected size.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
Bibliography：
[1] Clive James.2015 whole world biotechnology/genetically modified crops commercialized development situation [J] China is raw
Thing engineering magazine, 2016,36 (4):1-11.(James C.Global biotechnology/GM crops
commercialization development trend in 2015[J].China Biotechnology 2016,36
(4):1-11(in chinese))；
[2] Cui Ningbo, Zhang Zhengyan genetically engineered soybeans are studied and application progress [J] northwests agricultural journal, 2016,25 (8):
1111-1124.(Cui N B,Zhang Z Y.Research and application of transgenic soybean
[J].Acta Ageiculturae Boreali-occidentalis Sinia2016,25(8):1111-1124.)；
[3] progress [J] Chinese biological engineering magazines of Guo Bin, Qi Yang, Wei Ya brightness transgenic plant detection technologies,
2010,30 (2)：120-126.(Guo B,Qi Y,Wie Y H.Progress in the research of transgenic
plant testing[J].China Biotechnology2010,30(2):120-126.)；
[4] research method of Liu Bei foreign genes insertion point flanking sequence and progress [J] agriculturals and technology, 2012
(4)：97.(Wang X B,Jiang L X,2,Wei L,et al.Integration and Insertion Site of
EPSPs Gene on the Soybean Genome in Genetically Modified Glyphosate-Resistant
Soybean[J].ACTA AGRONOMICA SINICA 2010,36(3):365-375.)；
[5]Fulton T M,Chunwongse J,Tanksley S D.Microprep protocol for
extraction of DNA from tomato and other herbaceous plants[J].Plant Molecular
Biology Reporter,1995,13(3):207-209。
SEQUENCE LISTING
<110>Jilin Academy of Agricultural Science
<120>A kind of genetically engineered soybean WH8013 transformation event foreigns insertion vector right side flap sequence
<160> 7
<170> PatentIn version 3.3
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ttggtaagct ccgtcatcga aac 23
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atgcggacgg gaaacatatc ag 22
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gaatactgaa gagctcggtg ttgc 24
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gttccctcag atgtgacttc ggaaggggat actttcccat tcgtggatcg ctttcaagta 180
aaggaaggtg tttttttggt atattcctca aatgactttg gaaaagaagg tacggactac 240
tttacttata ctggtagtgg tggaaatgag gttcacatct cgggcacctc ttcagaagca 300
ggaataaaac cgcagtttat tgaaacttgc catccaaaac atcttaagcg gggaacaaaa 360
gagcaggaag atataaatag tagtacctca aagaaaagtg cagttatcaa caatttttcg 420
ggtgaaaaaa caccaaatcc aaggccacag agttccaaca tttcagaaag agagacgtat 480
gtcggaatat tgaacgtcaa atgtaaaaat aagaactcat cgaaaatacg aagtgaaaaa 540
ttggtaagct ccgtcatcga aacaaagcat acgccaggat tggcatctat tttatcgaaa 600
gaaggcacta catatccgaa taatgcggac gggaaacata tcagtatcgt gaatccatcc 660
tcaaaaatat atcattcatc ccataaacag attgttaaaa cgcctatccc taagagtggc 720
ctttctccaa ttgagagatg ccctttcaat ggtcaaaata ttaaatgcta ctcaccaaga 780
ccactagatc atgaaagtcc ccaacgtgat ttcaataata actttcagct gagaatactg 840
aagagctcgg tgttgcaaag gagacaatca acacagaata gttgaggtga ccagctcgaa 900
tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg 960
tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat 1020
gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 1080
ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 1140
gtcatctatg ttactagatc gggaattaaa ctatcagttc gcgagagtag atttgtatgt 1200
tgtaattttt tgtccctatt tgagtatgca tgtacactgg tagtctcttt tcagtggttt 1260
agtaggccgc taaagtaacg atgctgtgat aaggaactgt tttttccttc ttccccccat 1320
ttttatagtc agaaccctgt taattgttca tcctgggccc ctggctgagt gaaactaagc 1380
ttccatgtta gattgaacaa gcaattgcaa agcttgtatt attattatta ttattatttt 1440
ttttttttta cgtgggttac ctaccatctt catacatatc cgcacccggt tcaacgattt 1500
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ccctggctga gtgaaactaa gcttccatgt tagattgaac aagcaattgc aaagcttgta 240
ttattattat tattattatt tttttttttt tacgtgggtt acctaccatc ttcatacata 300
tccgcacccg gttcaacgat ttaaaaacac aagttgtatt ggatgtgata taaattgctc 360
aaaattctgt gtataatcta aatttaagtc atgtggcagt gatatatatt ttttttttaa 420
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Claims (10)
- A kind of 1. right side flap sequence of genetically engineered soybean WH8013 transformation event foreign insertion vectors, it is characterised in that its nucleosides Acid sequence such as SEQ ID NO：Shown in 4.
- 2. application of the right side flap sequence described in claim 1 in genetically engineered soybean is detected, it is characterised in that the transgenosis is big PCHAL1 T-DNA fragments are inserted at the 21177109th nucleotides of No. 8 chromosome of beans；Preferably, the transgenosis is big Beans are WH8013 or the genetically engineered soybean by its transformation.
- 3. application according to claim 2, it is characterised in that right side flap sequences Design is used for according to claim 1 Detect the PCR primer pair or hybridization probe of the genetically engineered soybean.
- 4. the PCR primer pair of right side flap sequences Design according to claim 1, it is characterised in that the nucleosides of the primer pair Acid sequence is respectively such as SEQ ID NO：6 and SEQ ID NO：Shown in 7.
- 5. a kind of PCR detection kit, it is characterised in that the kit includes primer pair described in claim 4, and other Reagent.
- 6. kit according to claim 5, it is characterised in that other described reagents include archaeal dna polymerase, dNTPs, One or more in PCR reaction buffers, positive reference substance and negative controls.
- 7. kit according to claim 6, it is characterised in that the archaeal dna polymerase be selected from Taq, Bst, Vent, Phi29、Pfu、Tru、Tth、Tl1、Tac、Tne、Tma、Tih、Tf1、Pwo、Kod、Sac、Sso、Poc、Pab、Mth、Pho、 One or more in ES4DNA polymerases, Klenow fragments；Preferably, the archaeal dna polymerase is Taq archaeal dna polymerases；More Preferably, the Taq archaeal dna polymerases are thermal starting Taq archaeal dna polymerases.
- 8. kit according to claim 5, it is characterised in that other described reagents include the premixed liquid of PCR reactions, institute Premixed liquid is stated as archaeal dna polymerase, the mixture of dNTPs and PCR reaction buffers.
- 9. the application method of any one of primer pair described in claim 4 or claim 5~8 kit, it is characterised in that Methods described includes：(1) using the DNA of soybean sample to be measured as template, any one of usage right requirement 4 described primer pair or claim 5~8 Primer pair and other reagents in the kit enter performing PCR amplification；(2) judged according to pcr amplification product on No. 8 chromosome of the soybean to be measured at 21177109th nucleotides whether Insert pCHAL1 T-DNA fragments.
- 10. according to the method for claim 9, it is characterised in that the annealing temperature of the PCR reactions is 49~51 DEG C, is followed Number of rings is 28~32 times；Preferably, the annealing temperature is 50 DEG C, and the period is 30 times.
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| CN112553370A (en) * | 2020-12-30 | 2021-03-26 | 吉林省农业科学院 | Exogenous inserted fragment flanking sequence of salt-tolerant transgenic soybean event E8A7027 and application |
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| CN108239637A (en) * | 2018-02-03 | 2018-07-03 | 吉林省农业科学院 | Disease-resistant transgenic soybean event B5B8127-3 external source Insert Fragment flanking sequences and its application |
| CN112553370A (en) * | 2020-12-30 | 2021-03-26 | 吉林省农业科学院 | Exogenous inserted fragment flanking sequence of salt-tolerant transgenic soybean event E8A7027 and application |
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