CN107236733A - The transformation event foreign insertion vector flanking sequences of genetically engineered soybean W82 HAL1 8062 and its application - Google Patents
The transformation event foreign insertion vector flanking sequences of genetically engineered soybean W82 HAL1 8062 and its application Download PDFInfo
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Abstract
The invention discloses the transformation event foreign insertion vector left side flap sequences of genetically engineered soybean W82 HAL1 8062 and right side flap sequence, its sequence such as SEQ ID NO：4 or SEQ ID NO：Shown in 8.The transformation event foreign insertion vector flanking sequences of genetically engineered soybean W82 HAL1 8062 of the present invention can be used for specific qualitative detection soybean and whether Related product contains the transformation events of W82 HAL1 8062.Present invention sequencing first discloses the transformation event foreigns of genetically engineered soybean W82 HAL1 8062 insertion flanking sequence, establishes the specificity of transformant qualitative PCR detection methods of genetically engineered soybean W82 HAL1 8062, this method is sensitive, accurately, simply, reliably, with wide market prospects.
Description
Technical field
The present invention relates to genetically engineered soybean field, in particular to genetically engineered soybean W82-HAL1-8062 transformation events
Exogenous insertion vector flanking sequence and its application.
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 developed in a healthy way is ensured, and detection GMOs technology is one of key technology that bio-safety is evaluated[3],
Authentication for transfer-gen plant is significant, and helps to exist to the expression mechanism and foreign gene of foreign gene
The influence produced in acceptor gene group is studied, so that the biological safety helped for transfer-gen plant makes assessment[4]。
HAL1 genes are related to salt tolerant genes 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 genetically engineered soybean W82-HAL1-8062 transformation event foreigns insertion vector left side
Flanking sequence, the sequence includes the T-DNA on pCHAL1 carriers and the soybean gene group sequence on the left of the T-DNA insertion points
Row, the sequence is the genetically engineered soybean for the T-DNA for inserting pCHAL1 on No. 1 chromosome at the 49468395th nucleotides
Specific fragment, can specifically indicate above-mentioned genetically engineered soybean.
The second object of the present invention is to provide the genetically engineered soybean W82-HAL1-8062 transformation event foreigns insertion vector right side
Flanking sequence, the sequence includes the T-DNA right side flaps sequence on pCHAL1 carriers and the soybean on the right side of the T-DNA insertion points
Genome sequence, the DNA fragmentation is turn for the T-DNA for inserting pCHAL1 on No. 1 chromosome at the 49468395th nucleotides
The specific fragment of transgenic soybean, can specifically indicate above-mentioned genetically engineered soybean.
The third object of the present invention is to provide above-mentioned left side flap sequence or right side flap sequence in detection genetically engineered soybean
Application, can quickly and accurately detect sample whether to come from No. 1 chromosome at the 49468395th nucleotides and insert
The genetically engineered soybean of pCHAL1 T-DNA fragments, especially W82-HAL1-8062 or by W82-HAL1-8062 breedings
Genetically engineered soybean.
The fourth object of the present invention is PCR primer pair of the offer according to above-mentioned left side flap sequences Design, passes through above-mentioned PCR
Primer pair can quickly and accurately detect what is whether inserted at the 49468395th nucleotides on No. 1 chromosome of testing sample
PCHAL1 T-DNA, has the advantages that easy to detect, specific good, amplification efficiency is high and accuracy is good.
The fifth object of the present invention is PCR primer pair of the offer according to above-mentioned right side flap sequences Design, passes through above-mentioned PCR
Primer pair can quickly and accurately detect whether testing sample inserts pCHAL1 T-DNA, with easy to detect, specificity
Good, amplification efficiency height and the good advantage of accuracy.
The sixth object of the present invention is to provide a kind of kit for PCR, the kit by above-mentioned primer pair and
Other auxiliary reagents that PCR is used are integrated, and are had the advantages that easy to use.
The seventh object of the present invention is to provide above-mentioned primer pair or the application method of kit, and methods described passes through simple
PCR be T-DNA that whether detectable sample inserts pCHAL1, it is good with easy to detect, specific good and accuracy
Advantage.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
Genetically engineered soybean W82-HAL1-8062 transformation event foreign insertion vector left side flap sequences, the left side flap sequence
Nucleotide sequence such as SEQ ID NO：Shown in 4.
The present invention is set up on the genetically engineered soybean W82-HAL1-8062 bases provided in Jilin Academy of Agricultural Science.Turn
Transgenic soybean W82-HAL1-8062 is by the plasmid pCHAL1 with HAL1 genes by agriculture bacillus mediated soybean cotyledon node method
T-DNA be inserted partially on the genome of soybean WILLIAMS-DARLING Ton 82 and obtain, it has preferable salt tolerance, can be saline and alkaline
Ground is survived, with preferable application and Breeding value.But before making the present invention, still Nobody Knows T-DNA specific insertion point,
The method that can effectively identify the genetically engineered soybean can not more be set up.
Well-designed 5 ' the SP1 of the present invention, 5 ' SP2 and 5 ' SP3 sequences, with reference to the Genome Walking of TakaRa companies
Kit kits, the flanking sequence on genetically engineered soybean HAL1-W82 on the left of T-DNA is obtained by TAIL-PCR, by be sequenced with
And compare, the final sequence for determining the DNA fragmentation on the left of genetically engineered soybean W82-HAL1-8062 transformation event foreign insertion vectors
Row, and T-DNA insertion point for the 49468395th nucleotides on No. 1 chromosome at.The area of foreign gene insertion
Domain is noncoding region.External source T-DNA integrate region and the stable expression and the generation of gene silencing of foreign gene has very
Close relationship, this T-DNA is inserted into gene noncoding region, it is to avoid what the insertion of foreign gene was caused to endogenous gene expression
Influence.Therefore, genetically engineered soybean HAL1-W82 of the present invention is suitable transgenic line, available for follow-up breeding, tool
There is preferable application prospect.
The above-mentioned genetically engineered soybean W82-HAL1-8062 transformation event foreigns insertion vector of the present invention and left side flap sequence are contained
The soybean genomic sequence covered on the left of pCHAL1 plasmid fractions T-DNA and the T-DNA insert divisions, can specifically be characterized in
The genetically engineered soybean of pCHAL1 T-DNA sequences is inserted at 49468395th nucleotides of No. 1 chromosome.Based on the sequence
Row, can specifically detect whether testing sample is derived from above-mentioned genetically engineered soybean, detection and germplasm in genetically engineered soybean
The identification aspect of resource is significant and is widely applied prospect.
The present invention also provides genetically engineered soybean W82-HAL1-8062 transformation event foreign insertion vector right side flap sequences, institute
State the nucleotide sequence such as SEQ ID NO of right side flap sequence：Shown in 8.
Well-designed 3 ' the SP1 of the present invention, 3 ' SP2 and 3 ' SP3 sequences, with reference to the Genome Walking of TakaRa companies
Kit kits, T-DNA right side flap sequences on genetically engineered soybean HAL1-W82 are obtained by TAIL-PCR, by being sequenced and comparing
It is right, the final sequence for determining the DNA fragmentation on the right side of genetically engineered soybean W82-HAL1-8062 transformation event foreign insertion vectors.
Gene can also be special on the right side of the above-mentioned genetically engineered soybean W82-HAL1-8062 transformation event foreigns insertion vector of the present invention
The transgenosis for being characterized in the T-DNA sequences that pCHAL1 is inserted at the 49468395th nucleotides of No. 1 chromosome different in naturely is big
Beans.Based on the sequence, it can specifically detect whether testing sample is derived from above-mentioned genetically engineered soybean, in the inspection of genetically engineered soybean
Survey and the identification aspect of germ plasm resource is significant.
The present invention also provides the application of above-mentioned left side flap sequence or right side flap sequence in detection genetically engineered soybean, described turn
PCHAL1 T-DNA fragments are inserted on No. 1 chromosome of transgenic soybean at 49468395th nucleotides；Preferably, it is described
Genetically engineered soybean is W82-HAL1-8062 or the genetically engineered soybean by W82-HAL1-8062 breedings.
In some embodiments, the application includes being used to examine according to above-mentioned left side flap sequence or right side flap sequences Design
Survey the PCR primer pair or hybridization probe of the genetically engineered soybean.
The present invention also provides the PCR primer pair according to above-mentioned left side flap sequences Design, the nucleotide sequence of the primer pair
Such as SEQ ID NO：10 and SEQ ID NO：Shown in 11.
A design in the above-mentioned primer pair of the present invention is on T-DNA BAR genes, and another design is on the left of T-DNA
Soybean gene group on, therefore, amplification when, be only inserted into T-DNA at the 49468395th nucleotides of No. 1 chromosome
Genetically engineered soybean could expand for template and obtain purpose fragment, using other genetically engineered soybeans or Non-transgenic soybean as template
Acquisition purpose fragment can not be expanded, so as to easily judge to detect whether object is that T-DNA is inserted into the of No. 1 chromosome
Genetically engineered soybean at 49468395 nucleotides.In addition, rear of the above-mentioned primer pair of the invention by well-designed and screening
Obtain, have the advantages that specific good, detection efficiency is high and accuracy is good.Therefore, primer pair of the present invention turns base to raising
Because the determination rates of material, the management of transgenic product and detection, and the identification of germ plasm resource have great importance.
The present invention also provides the PCR primer pair according to above-mentioned right side flap sequences Design, the nucleotide sequence of the primer pair
Such as SEQ ID NO：12 and SEQ ID NO：Shown in 13.
A design in the above-mentioned primer pair of the present invention is on T-DNA HAL1 genes, and another design is on the right side of T-DNA
Soybean gene group on, therefore, amplification when, be only inserted into T-DNA at the 49468395th nucleotides of No. 1 chromosome
Genetically engineered soybean could expand for template and obtain purpose fragment, using other genetically engineered soybeans or Non-transgenic soybean as template
Acquisition purpose fragment can not be expanded, so as to easily judge to detect whether object is that T-DNA is inserted into the of No. 1 chromosome
Genetically engineered soybean at 49468395 nucleotides.In addition, rear of the above-mentioned primer pair of the invention by well-designed and screening
Obtain, have the advantages that specific good, detection efficiency is high and accuracy is good.Therefore, primer pair of the present invention turns base to raising
Because the determination rates of material, the management of transgenic product and detection, and the identification of germ plasm resource have great importance.
The present invention also provides a kind of kit for PCR, and the kit includes above-mentioned primer pair, and other examinations
Agent.
Mentioned reagent box of the present invention integrates above-mentioned primer pair with other auxiliary reagents that PCR is used, with making
Use convenient advantage.
In some embodiments, other described reagents are right including water, archaeal dna polymerase, dNTPs, PCR buffer solution, the positive
According to the one or more in product and negative controls；Preferably, 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 gather
One or more in synthase, Klenow fragments；It is highly preferred that the archaeal dna polymerase is Taq archaeal dna polymerases；Most preferably,
The Taq archaeal dna polymerases are thermal starting Taq archaeal dna polymerases.
The present invention also provides above-mentioned PCR primer pair or the application method of kit, and methods described is included with testing sample
DNA is that template, above-mentioned primer pair are that upstream and downstream primer enters performing PCR amplification, and No. 1 of the sample is judged according to pcr amplification product
Whether pCHAL1 T-DNA fragment is inserted at the 49468395th nucleotides on chromosome.
The above-mentioned application method of the present invention passes through on No. 1 chromosome that simple PCR method is detectable sample the
The HAL1 genes whether inserted at 49468395 nucleotides, with good excellent of easy to detect, specific good and accuracy
Point.
In some embodiments, the annealing temperature of the PCR reactions is 50-54 DEG C, and period is 30-35；Preferably,
The annealing temperature of the PCR reactions is 52 DEG C, and period is 35.
In some embodiments, the annealing temperature of the PCR reactions is 48-50 DEG C, and period is 30-35, it is preferable that
The annealing temperature of the PCR reactions is 49 DEG C, and period is 35.
Compared with prior art, beneficial effects of the present invention are：
The present invention relates to genetically engineered soybean W82-HAL1-8062, the 49468395th on No. 1 chromosome of the soybean
The T-DNA sequences inserted at nucleotides on pCHAL1, so as to obtain HAL1 genes, the genetically engineered soybean can Salt And Alkali Tolerance,
Salt-soda soil is survived, with good application prospect.The present invention is obtained outside genetically engineered soybean W82-HAL1-8062 transformation events first
Left side flap sequence and right side flap sequence are inserted in source, devise corresponding PCR primer and establish PCR method for detecting specificity, energy
Whether enough testing samples of detection exactly insert pCHAL1 T- at the 49468395th nucleotides on No. 1 chromosome
The genetically engineered soybean of DNA sequence dna, this method is sensitive, accurately, simply, reliably, with wide market prospects, to improving transgenosis
The determination rates of material, the management of transgenic product and detection and the identification of germ plasm resource have great importance.
Secondly, above-mentioned primer pair of the invention is finally obtained through well-designed and repeated screening side, separately design in T-DNA and
In the soybean gene group of T-DNA flanks, turning at the 49468395th nucleotides of No. 1 chromosome is only inserted into T-DNA
Transgenic soybean could expand for template and obtain purpose fragment, can not as template using other genetically engineered soybeans or Non-transgenic soybean
Amplification obtains purpose fragment, so as to easily judge to detect whether object is that T-DNA is inserted into the of No. 1 chromosome
Genetically engineered soybean at 49468395 nucleotides.In addition, above-mentioned primer pair of the invention also has specific good, detection efficiency high
The good advantage with accuracy.
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 accompanying drawing used required in embodiment or description of the prior art is briefly described, 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 is pCHAL1 plasmid maps；
Fig. 2 is left margin TAIL-PCR electrophoresis results, wherein, M1：λ-HindⅢdigest；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：It is just right
According to 1st 2nd 3rd；M2：DL2000；
Fig. 3 is left margin TAIL-PCR sequencing result, wherein, plus frame portion is divided into soybean genomic sequence, overstriking italic
Part is does not match sequence, with the Bar gene orders that dash area is T-DNA；
Fig. 4 is right margin TAIL-PCR electrophoresis results, wherein, M1：λ-HindⅢdigest；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：It is just right
According to 1st 2nd 3rd；M2：DL 2000；
Fig. 5 is right margin TAIL-PCR sequencing result, wherein, plus frame portion is divided into T-DNA HAL1 genes, overstriking italic
Part is does not match sequence, and band dash area is soybean genomic sequence；
Fig. 6 is soybean gene group Chr01:49468395..49469837 sequence；
Fig. 7 is genetically engineered soybean F1H6018 right boundary primer location figures；
Fig. 8 is genetically engineered soybean HAL1-W82 specific PCR testing result (marker tape bp), wherein, 1-5 swimming lanes are the right
The F of boundary's primer 3 ' and 3 ' R amplification (1,2,3 is positive material, and 4 be negative control, and 5 be plasmid)；6-10 swimming lanes are the left side
The F of boundary's primer 5 ' and 5 ' R amplification (6,7,8 be positive material, and 9 be negative control, and 10 be plasmid)；M is 100BP
marker。
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 advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
Genetically engineered soybean W82-HAL1-8062 left margin flanking sequences are obtained by the following method：
1st, genetically engineered soybean W82-HAL1-8062 (the genetically engineered soybean W82-HAL1-8062 acquisitions of field planting are taken
From Jilin Academy of Agricultural Science) blade in -80 DEG C preservation, the genetically engineered soybean W82-HAL1-8062 is with soybean William
Nurse this 82 be acceptor material, using plasmid pCHAL1 as conversion carrier, by agriculture bacillus mediated soybean cotyledon node method for transformation obtain,
The collection of illustrative plates of the plasmid pCHAL1 is referring to Figure of description Fig. 1.
2nd, soybean leaves DNA is extracted using CTAB methods, with the purity of the DNA sample of NanoDrop Detection and Extraction and dense
Degree, specific method is referring to bibliography 5.
3rd, 3 specific primers are designed according to the bar genes on pCHAL1 plasmid maps, specific primer sequence is referring to table 1.
The left margin specific primer of table 1
4th, the transgenic soybean gene group DNA using 1 μ l (500ng) is template, with 5 ' SP1,5 ' SP2,5 ' SP3 and TaKaRa
AP1, AP2 and AP3, AP4 in Genome Walking Kit (Code No.6108) kit, and ck primers, are pressed
TaKaRaGenome Walking Kit (Code No.6108) kit specification is carried out 5 times, and 3 wheel TAIL-PCR are anti-every time
Should.
To PCR primer carry out electrophoresis, electrophoresis result as shown in Figure of description Fig. 2, Fig. 2 show 4 degenerate primer AP1,
AP2, AP3, AP4 the 3rd wheel PCR reactions (3,6,9,12 swimming lane) obtain providing positive control (15 swimming lane) clearly than kit
Specific band.
5th, the amplified production band (9 swimming lane) for 5 ' the AP3-3rd about 1.0kbp being of moderate size is chosen, Takara is used
MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No.9762) gel extraction specificity expands
Increase product band.
6th, glue reclaim product is connected with pMDTM19-TVector (Code No.6013), conversion to E.coli
In Competent Cells JM109 (Code No.9052).
7th, the positive single bacterium colony of picking, is sequenced, and finally obtains 5 ' the unknown flanking sequences in end, sequencing result such as SEQ ID
NO：(referring to Figure of description Fig. 3) shown in 4.
Embodiment 2
Genetically engineered soybean W82-HAL1-8062 right margin flanking sequences are obtained by the following method：
1st, 3 specific primers are designed according to the HAL1 genes on pCHAL1 plasmid maps, specific primer sequence is referring to table
2。
The right margin specific primer of table 2
2nd, using 1 μ l (500ng) embodiment 1 prepare genomic DNA as template, with 3 ' SP1,3 ' SP2,3 ' SP3 with
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.
Electrophoresis is carried out to PCR primer, electrophoresis result is as shown in Figure 4.Fig. 4 shows primer AP1, AP2, AP3 the 3rd wheel PCR
Reaction (3,6,9 swimming lane) obtains providing positive control (15 swimming lane) clearly specific band, and molecular weight than kit
Size is similar.
3rd, the amplified production band (9 swimming lane) with 3 ' AP3-3rd of 5 ' same primers is chosen for convenience of sequencing, used
Takara MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No.9762), gel extraction
It is sequenced, sequencing result such as SEQ ID NO：(see Figure of description Fig. 5) shown in 8.
Embodiment 3
Integration sites of the external source T-DNA in soybean gene group is determined in accordance with the following methods：
According to the flanking sequence sequencing result of acquisition in soybase websites (https://www.soybase.org/
GlycineBlastPages/ soybean gene group) is compared, determines that T-DNA inserts soybean gene No. 1 chromosome noncoding region of group
At 49468395 bases.
Such as Fig. 6 soybean gene group Gm01_49468395..49469837 sequences (SEQ ID NO：9) shown in, band frame is shown
First half and left margin sequencing result band frame portion sub-sequence match (Fig. 3), with the latter half shown by dash area and
Right margin sequencing result adds the sequences match (Fig. 5) of frame portion point, and these results illustrate the T-DNA of left and right avris flank sequence positioning
Integration region in soybean gene group is completely the same, and this transgenic event is single copy transgenic event.T-DNA is being incorporated into
During soybean gene group, carrier right boundary sequence is deleted, and left margin is filled with the core in 8bp one section of unknown source
Nucleotide sequence, right margin is filled with the nucleotide sequence in 4bp one section of unknown source.
The genetically engineered soybean specific PCR of embodiment 4 is detected
1st, PCR primer pair is designed according to flanking sequence sequencing result：Design PCR primer pair, its one end in T-DNA sequences,
The other end is in soybean gene group：Such as 5 ' F sense primers are on left margin genome, and 5 ' R anti-sense primers are on T-DNA, on 3 ' F
Primer is swum on T-DNA, 3 ' R anti-sense primers are on the right (Fig. 7) on boundary's genome.Specific primer sequence is referring to table 3.
Table 3：5 ' and 3 ' end primers
2nd, enter performing PCR using above-mentioned primer to detect, wherein 5 ' reaction conditions are：
1st step：94℃ 2min
2nd step：94℃ 30s
3rd step：52℃ 30s
4th step：72℃ 1min
5th step：72℃ 5min
6th step：16℃ 1h
2nd~4 step is circulated 35 times.
3 ' reaction conditions are：
1st step：94℃ 2min
2nd step：94℃ 30s
3rd step：49℃ 30s
4th step：72℃ 1min
5th step：72℃ 5min
6th step：16℃ 1h
2nd~4 step is circulated 35 times.
3rd, amplified production is detected using 2% Ago-Gel, can not be expanded to band in plasmid and negative-type, only
Transgenic positive material obtains the fragment (Fig. 8) 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, or 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] the global biotechnologys of Clive James.2015/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 the agriculture journal in application progress [J] northwests, 2016,25 (8):
1111-1124.(Cui N B,Zhang Z Y.Research and application of transgenic soybean
[J].Acta Ageiculturae Boreali-occidentalis Sinia 2016,25(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 Biotechnology 2010,30(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>Genetically engineered soybean W82-HAL1-8062 transformation event foreign insertion vector flanking sequences and its application
<160> 13
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ccagagaacg tcctctttca gtgattttgc catttcatta gctttgttaa atgatgttta 180
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tcgtaaatag cctttaaaag tgataactta atgaggtgaa aaagtcagag aatcaagtca 420
ataataataa agaatgagat gagatgggca aagttttaga tctttagcca gttgtagtag 480
cctagagcta actagacttt tgttctggaa tgtattattt tggctggatt ccttagacag 540
tagatagcat gacacctcca cttctataga cattgctgaa tggaaaaaaa ttataagcag 600
agtaccgaat aaaagaattg gtcccactga gtaccagaat atattgtggt gtaaacaaat 660
tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga attaacgccg 720
aattaattcg ggggatctgg attttagtac tggattttgg ttttaggaat tagaaatttt 780
attgatagaa gtattttaca aatacaaata catactaagg gtttcttata tgctcaacac 840
atgagcgaaa ccctatagga accctaattc ccttatctgg gaactactca cacattatta 900
tggagaaact cgagtcaaat ctcggtgacg ggcaggaccg gacggggcgg taccggcagg 960
ctgaagtcca gctgccagaa acccacgtca tgccagttcc cgtgcttgaa gccggccgcc 1020
cgcagcatgc cgcggggggc atatccgagc gcctcgtgca tgcgcacgct cgggtcgttg 1080
ggcagcccga tgacagcgac cacgctcttg aagccctgtg cctccaggga cttcagcagg 1140
tgggtgtaga gcgtggagcc cagtcccgtc cgctggtggc ggggggagac gtacacggtc 1200
gactcggccg tccagtcgta ggcgttgcgt gccttccagg ggcccgcgta ggcgatgccg 1260
gcgacctcgc cgtccacctc ggcgacgagc cagggatagc gctcccgcag acggacgagg 1320
tcgtccgtcc actcctgcgg ttcctgcggc tcggtacgga agttgaccgt gcttgtctcg 1380
atgtagtggt tgacgatggt gcagaccgcc ggcatgtccg cctcggtggc acggcggatg 1440
tcggccgggc gtcgttctgg gctcat 1466
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
acctcaaaga aaagtgcagt tatc 24
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
cgaaacaaag catacgccag gatt 24
<210> 7
<211> 24
<212> DNA
<213>Artificial sequence
<400> 7
taaaacgcct atccctaaga gtgg 24
<210> 8
<211> 1998
<212> DNA
<213>Artificial sequence
<400> 8
atgcatttca aagatttagg attgcatgac tacactctca aaaacttgat gtatgagaat 60
aattgctgta aattttatga tgccgtggat gaaaacaaca tctcatatgt tttaaaattt 120
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 ctatcagtgt ttgaaaaaga gttttttcag 1200
aaacactgat taaaaataaa aggctatatc ttaattaaca agtttttaga aaaactgatt 1260
aacatttaaa aaaaaaatag tttccgtatt ttaaaagaat aayaattttt gttccatcct 1320
taattttgca catttatttt ttatatttta attaattaat tatttcttgt tgtacctcaa 1380
atgatatgtt agatttggtg tttaattgaa tcacggtaaa aagaaataaa atataaataa 1440
aattaagrat cggacaaaaa ttataaaaaa aatttaaata tgaagagtaa rattaaaaaa 1500
aaggactaat gggaattaag aattaaagag gatcraaatg aaatttaagt agcccaaata 1560
ggaagtatag tgtttcgtat aatttaaaat aaaatgtatt tccataattt ttttttaaga 1620
tccttaacat ttgcttgaaa gaatacacac aaatacrata taaaataaaa aagaatggat 1680
atgttaaaga aatattgaag gaatcatarg aagaaaatat aaaattattt tgttttctta 1740
ataatttgcc tccttgattg ccatccaatc ttttagaatg gcctatttga aattttggca 1800
ttgtggaata tagtttggty cctgtaatag cctcttatga tcggtcgytg ttagaagatt 1860
gatctcatat catgtcatta atcataattt taataatagg ttccatccat atgtgaaaat 1920
tttgttggtg gataaattat aagttaaata gtttttaaaa ttttgagrtc tatcccaatt 1980
ctaaaaaaaa attacgga 1998
<210> 9
<211> 1442
<212> DNA
<213> Glycine max
<400> 9
cgacggatag tgggggagaa agagaaaccg aagatgatta tgtactatgg ttattatata 60
tgtacaattg taacttcact ctattgttaa acgaattaca gtttaccctt tgtaccactt 120
ccagagaacg tcctctttca gtgattttgc catttcatta gctttgttaa atgatgttta 180
ctttgctgga acttttaaat gggaagaaat cgaaaattat ggaaggacta atagtaacaa 240
tgaaaaagag gtaagtaaaa tcattaacca ttatcatcat cttcttctgt tagttaatca 300
cgtagcagcc atcttggttc atttaatcac atgggcagct ggagactgcc tatacatgtt 360
tcgtaaatag cctttaaaag tgataactta atgaggtgaa aaagtcagag aatcaagtca 420
ataataataa agaatgagat gagatgggca aagttttaga tctttagcca gttgtagtag 480
cctagagcta actagacttt tgttctggaa tgtattattt tggctggatt ccttagacag 540
tagatagcat gacacctcca cttctataga cattgctgaa tggaaaaaaa ttataagcag 600
agtaccgaat aaaagaattg gtcccactga agagtttttt cagaaacact gattaaaaat 660
aaaaggctat atcttaatta acaagttttt agaaaaactg attaacattt aaaaaaaaaa 720
tagtttccgt attttaaaag aataacaatt tttgttccat ccttaatttt gcacatttat 780
tttttatatt ttaattaatt aattatttct tgttgtacct caaatgatat gttagatttg 840
gtgtttaatt gaatcacggt aaaaagaaat aaaatataaa taaaattaag aatcggacaa 900
aaattataaa aaaaatttaa atatgaagag taaaattaaa aaaaaggact aatgggaatt 960
aagaattaaa gaggatcaaa atgaaattta agtagcccaa ataggaagta tagtgtttcg 1020
tataatttaa aataaaatgt atttccataa tttttttttt aagatcctta acatttgctt 1080
gaaagaatac acacaaatac aatataaaat aaaaaagaat ggatatgtta aagaaatatt 1140
gaaggaatca taagaagaaa atataaaatt attttgtttt cttaataatt tgcctccttg 1200
attgccatcc aatcttttag aatggcctat ttgaaatttt ggcattgtgg aatatagttt 1260
ggttcctgta atagcctctt atgatcggtc gttgttagaa gattgatctc atatcatgtc 1320
attaatcata attttaataa taggttccat ccatatgtga aaattttgtt ggtggataaa 1380
ttataagtta aatagttttt aaaattttga ggtctatccc aattctaaaa aaaaattacg 1440
ga 1442
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
gtagcagcca tcttggttca 20
<210> 11
<211> 19
<212> DNA
<213>Artificial sequence
<400> 11
gcaacgccta cgactggac 19
<210> 12
<211> 18
<212> DNA
<213>Artificial sequence
<400> 12
gaaggcacta catatccg 18
<210> 13
<211> 19
<212> DNA
<213>Artificial sequence
<400> 13
ccgtgattca attaaacac 19
Claims (10)
1. genetically engineered soybean W82-HAL1-8062 transformation event foreign insertion vector left side flap sequences, it is characterised in that the left side
The nucleotide sequence of flanking sequence such as SEQ ID NO：Shown in 4.
2. genetically engineered soybean W82-HAL1-8062 transformation event foreign insertion vector right side flap sequences, it is characterised in that the right side
The nucleotide sequence of flanking sequence such as SEQ ID NO：Shown in 8.
3. the right side flap sequence described in left side flap sequence or claim 2 described in claim 1 is in detection genetically engineered soybean
Application, pCHAL1 T-DNA pieces are inserted on No. 1 chromosome of the genetically engineered soybean at the 49468395th nucleotides
Section；Preferably, the genetically engineered soybean is W82-HAL1-8062 or the genetically engineered soybean by W82-HAL1-8062 breedings.
4. application according to claim 3, it is characterised in that left side flap sequence or right according to claim 1
It is required that the right side flap sequences Design described in 2 is used for the PCR primer pair or hybridization probe for detecting the genetically engineered soybean.
5. the PCR primer pair of left side flap sequences Design according to claim 1, it is characterised in that the nucleosides of the primer pair
Acid sequence such as SEQ ID NO：10 and SEQ ID NO：Shown in 11.
6. the PCR primer pair of right side flap sequences Design according to claim 2, it is characterised in that the nucleosides of the primer pair
Acid sequence such as SEQ ID NO：12 and SEQ ID NO：Shown in 13.
7. a kind of kit for PCR, it is characterised in that the kit includes drawing described in claim 5 or claim 6
Thing pair, and other reagents.
8. kit according to claim 7, it is characterised in that other described reagents include water, archaeal dna polymerase,
One or more in dNTPs, PCR buffer solution, positive reference substance and negative controls；Preferably, the archaeal dna polymerase is selected from
Taq、Bst、Vent、Phi29、Pfu、Tru、Tth、Tl1、Tac、Tne、Tma、Tih、Tf1、Pwo、Kod、Sac、Sso、Poc、
One or more in Pab, Mth, Pho, ES4DNA polymerase, Klenow fragments；It is highly preferred that the archaeal dna polymerase is Taq
Archaeal dna polymerase；Most preferably, the Taq archaeal dna polymerases are thermal starting Taq archaeal dna polymerases.
9. the application method of any one of any one of the claim 5-6 PCR primers pair or claim 7-8 kit,
Characterized in that, it is that upstream and downstream is drawn that methods described, which includes the primer pair by template of the DNA of testing sample, described in claim 6-7,
Thing enters performing PCR amplification, is judged according to pcr amplification product at the 49468395th nucleotides on No. 1 chromosome of the sample
Whether pCHAL1 T-DNA fragment is inserted.
10. method according to claim 9, it is characterised in that the annealing temperature of the PCR reactions is 50-54 DEG C, circulation
Number is 30-35, it is preferable that the annealing temperature of the PCR reactions is 52 DEG C, and period is 35；Or, what the PCR reacted moves back
Fiery temperature is 48-50 DEG C, and period is 30-35, it is preferable that the annealing temperature of the PCR reactions is 49 DEG C, and period is 35.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334600A (en) * | 2020-03-11 | 2020-06-26 | 南开大学 | Flanking sequence of transgenic soybean 8c-ox-2 exogenous insertion fragment for high nitrogen utilization, acquisition method and application |
| CN111876516A (en) * | 2020-08-03 | 2020-11-03 | 浙江新安化工集团股份有限公司 | Flanking sequence of exogenous insertion vector for transgenic soybean WYN341GmC transformation event and application thereof |
| CN111961741A (en) * | 2020-08-03 | 2020-11-20 | 浙江新安化工集团股份有限公司 | Flanking sequence of exogenous insertion vector for transgenic soybean WYN001GmC transformation event and application thereof |
| CN112375838A (en) * | 2020-11-19 | 2021-02-19 | 中国热带农业科学院热带生物技术研究所 | T-DNA flanking sequence of transgenic sugarcane SCAG2 and transformation event specificity identification method thereof |
| CN112391490A (en) * | 2020-11-19 | 2021-02-23 | 中国热带农业科学院热带生物技术研究所 | Exogenous insert flanking sequence of transgenic sugarcane event SCAG5 and strain specificity detection method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334600A (en) * | 2020-03-11 | 2020-06-26 | 南开大学 | Flanking sequence of transgenic soybean 8c-ox-2 exogenous insertion fragment for high nitrogen utilization, acquisition method and application |
| CN111876516A (en) * | 2020-08-03 | 2020-11-03 | 浙江新安化工集团股份有限公司 | Flanking sequence of exogenous insertion vector for transgenic soybean WYN341GmC transformation event and application thereof |
| CN111961741A (en) * | 2020-08-03 | 2020-11-20 | 浙江新安化工集团股份有限公司 | Flanking sequence of exogenous insertion vector for transgenic soybean WYN001GmC transformation event and application thereof |
| CN111961741B (en) * | 2020-08-03 | 2022-02-15 | 浙江新安化工集团股份有限公司 | Flanking sequence of exogenous insertion vector for transgenic soybean WYN001GmC transformation event and application thereof |
| CN111876516B (en) * | 2020-08-03 | 2022-02-22 | 浙江新安化工集团股份有限公司 | Flanking sequence of exogenous insertion vector for transgenic soybean WYN341GmC transformation event and application thereof |
| CN112375838A (en) * | 2020-11-19 | 2021-02-19 | 中国热带农业科学院热带生物技术研究所 | T-DNA flanking sequence of transgenic sugarcane SCAG2 and transformation event specificity identification method thereof |
| CN112391490A (en) * | 2020-11-19 | 2021-02-23 | 中国热带农业科学院热带生物技术研究所 | Exogenous insert flanking sequence of transgenic sugarcane event SCAG5 and strain specificity detection method thereof |
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