CN113106117A - Method for obtaining known TDNA flanking sequence inserted into genome site - Google Patents
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Abstract
The invention belongs to the technical field of transgenosis, and particularly discloses a method for obtaining a known TDNA flanking sequence inserted into a genome site. The invention obtains a transgenic plant by an agrobacterium transformation method, the part inserted into the plant genome is the part from LB to RB region on a transgenic vector, and the plant genome sequence information near the insertion position is obtained through the LB-RB region in the known TDNA flanking sequence, thereby obtaining the position information of the transgene inserted into the genome; in addition, the invention can obtain genome sequence information without complicated experimental process and massive sequencing analysis, can complete the enrichment of specific fragments through a PCR program, can analyze and obtain the information of insertion positions in a small amount of data of a generation of sequencing, is simple and efficient, and has certain application prospect.
Description
Technical Field
The invention belongs to the technical field of transgenosis, and particularly relates to a method for obtaining a known TDNA flanking sequence inserted into a genome site.
Background
In the transgenic detection process, the PCR technology is commonly used for detecting whether a target gene exists, southern hybridization is generally used for detecting the copy number of the target gene, the qPCR technology is used for detecting the transcription condition of the target gene, and the Tail PCR is used for identifying the insertion condition of the target gene in a genome and the like; by analyzing the inserted genome locus, people can judge different transgenic lines, the homozygous and heterozygous conditions of transgenes and can track the condition of target genes more easily, so that the identification of the inserted locus is necessary for the industrial application of transgenic plants.
According to the principle of the current Agrobacterium transformation technology, the insertion site of the foreign gene in the genome is random, and the flanking sequence of the foreign gene in the genome is unique in each transformation event, so that different transgenic strains can be distinguished by detecting the flanking sequence. At present, the literature reports a specific detection method of transgenic crop transformation events such as corn, rape, soybean, rice and the like, and the detection method provides reliable technical support for breeding, production, processing and sale of transgenic crops. In literature (liu dong bo, chen national jed, zha qian, etc.. exogenous T-DNA sequence analysis and qualitative detection of antiviral transgenic soybean events [ J ] soybean science, 2020(1)), authors performed Whole Genome Sequencing (WGS) on soybeans through Illumina Xten platform, analyzed the flanking sequences of the transgenic events in combination with bioinformatics analysis and PCR technique, and initially found the boundaries of the insert sequence T-DNA and its flanking sequences by whole genome sequencing and comparing with soybean reference genome (wm82.a2.v1) and transformation vector sequences, and the results demonstrated the effectiveness and stability of whole genome sequencing in identifying transgenic crop T-DNA insertions and flanking sequence regions. In the literature (Wenshou military, Denglihua, Xiaoguoshua cherry. transgenic rice EB7001S event specificity detection method establishment [ J ]. agricultural biotechnology report 2014,22(5):621 one 631.), the authors obtained the flanking sequence of the insertion site of the foreign gene in transgenic rice EB7001S by using high efficiency thermal asymmetric PCR (high efficiency thermal asymmetric PCR, high TAIL-PCR) and long distance PCR (10 ng-distance PCR, LD-PCR) amplification, the method has strong specificity, good stability and high sensitivity, and can still be detected by the common PCR method when the amount of EB7001S genomic DNA doped in the template is 0.1%.
The previous research methods provide a large amount of pcr experiments and high-throughput sequencing to analyze the insertion of exogenous T-DNA, and although the results are high in specificity and accuracy, the process is complex and consumes a large amount of manpower and material resources. In order to analyze the insertion condition of the transgenic expression vector in the plant genome and the influence on the plant genome after the transgenic expression vector is transferred into a plant, the research provides a novel analysis method, and the genome sequence information can be obtained without complicated experimental processes and massive sequencing analysis.
Disclosure of Invention
In view of the above problems in the prior art, it is an object of the present invention to provide a method for obtaining a genomic site into which a known TDNA flanking sequence is inserted. Obtaining a transgenic plant by an agrobacterium transformation method, wherein the part inserted into the plant genome is the part from LB to RB region on the transgenic vector, and obtaining the plant genome sequence information near the insertion position by the known LB-RB region in the TDNA flanking sequence, thereby obtaining the position information of the transgenic inserted genome.
The invention is realized by the following technical scheme:
a method for obtaining a genomic site for insertion of a flanking sequence of a known TDNA, comprising the steps of:
s1, transforming a vector containing a known TDNA flanking sequence into a plant by an agrobacterium-mediated method to obtain a transgenic positive seedling;
s2, finding a 4-base restriction enzyme cutting site and/or a 6-base restriction enzyme cutting site at the RB end of the known TDNA flanking sequence;
s3, extracting genome DNA of the transgenic positive seedling, and selecting restriction enzymes with 4-base enzyme cutting sites and/or 6-base enzyme cutting sites to perform enzyme cutting operation on the genome DNA to obtain gene fragments with different lengths;
s4, carrying out enzyme linking operation on the gene fragment by using DNA ligase to obtain a ring forming fragment; according to the right sequence of the enzyme cutting site, a pair of specific primers is designed to amplify the cyclized segment, and the fragments are reversely extended to two ends and connected into linear segments;
and S5, recovering and purifying the linear fragment, sending the linear fragment to a first generation sequencing, and comparing the obtained sequencing result with the genome data in a database to obtain the information of the genomic locus where the known TDNA flanking sequence is inserted.
Further, in step S2, the RB terminal sequence of the known TDNA flanking sequence is shown in SEQ ID NO. 2.
Further, in step S2, the 4-base cleavage site is a Tail cleavage site.
Further, in step S2, the 6-base cleavage site is a HindIII cleavage site.
Further, in step S3, the specific digestion reaction process of the Tail cleavage site is as follows: mu.L of genomic DNA, 1. mu.L of 10 Xdigestion buffer and 1. mu.L of Tail endonuclease were added, and the mixture was reacted at 65 ℃ for 4 hours, followed by heat inactivation at 80 ℃.
Further, in step S3, the Hind iii cleavage site is specifically cleaved as follows: mu.L of genomic DNA, 1. mu.L of 10 Xdigestion buffer, and 1. mu.L of Hind III endonuclease were added, and the mixture was reacted at 37 ℃ for 4 hours, followed by heat inactivation at 80 ℃.
Further, in step S4, the enzyme-linked reaction process is as follows: mu.L of the digested product, 1. mu. L T4 ligase and 1. mu.L of 10 Xligase buffer were added, and the mixture was reacted at 16 ℃ for 5 hours, followed by storage at 4 ℃.
Further, in step S4, the specific primer sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4.
In the invention, an enzyme cutting site is searched near the end positions of two arms at one side of a vector containing known TDNA flanking sequences, and if the enzyme cutting site is a 4-base enzyme cutting site, the enzyme cutting site is 44256bp must have a repeat; if it is a 6-base cleavage site, then 46Since 4096bp is inevitably repeated several times, the same cleavage site is present on the genome. Selecting a 4-base enzyme cutting site TaiI/MaeI [ acgt ] from the right arm sequence of the T-DNA]A 6-base restriction site HindIII [ aagctt ]]Then extracting the genome DNA of the transgenic plant,cleavage will result in three linear fragments: vector sequence, vector + genome, genome. And (3) connecting the two loops by using ligase to form a loop, wherein the expected loop contains a part of RB terminal sequence information of the known vector and a part of genome information near a desired insertion position, designing two primers near the known sequence, amplifying in two directions, amplifying the circular segment into linear segments with known sequences at two ends, and sequencing the amplified product for one generation to obtain the genome information near the transgene insertion position.
Compared with the prior art, the invention has the following advantages:
1) the invention obtains a transgenic plant by an agrobacterium transformation method, the part inserted into the plant genome is the part from LB to RB region on a transgenic vector, and the plant genome sequence information near the insertion position is obtained through the LB-RB region in the known TDNA flanking sequence, thereby obtaining the position information of the transgene inserted into the genome;
2) according to the invention, the genome sequence information can be obtained without complicated experimental processes and massive sequencing analysis, the enrichment of specific fragments can be completed through one-time PCR (polymerase chain reaction) procedure, and the information of insertion positions can be obtained by analysis in a small amount of data of first-generation sequencing, so that the method is simple and efficient and has a certain application prospect.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is an experimental schematic of the present invention;
FIG. 2 is a restriction enzyme electrophoresis detection diagram of the present invention; wherein Tail (1): using Tail restriction endonuclease to cut enzyme buffer and add ligase buffer at the same time; tail (2): using Tail restriction endonuclease, enzyme digestion buffer and ligase buffer are added in two steps; HindIII (1): using HindIII restriction endonuclease to cut enzyme buffer and add ligase buffer at the same time; HindIII (2): adding HindIII restriction endonuclease, enzyme digestion buffer and ligase buffer in two steps;
FIG. 3 is a graph of the present invention showing the results of sequencing in a spliced manner.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
The instruments and reagents used in the present invention are commercially available unless otherwise specified.
Example 1 preparation of samples of transgenic plants of Rice
A vector (pBWARE (I), the vector sequence is shown in SEQ ID NO:1) containing known TDNA flanking sequences is mediated and transformed into rice M294 through Agrobacterium tumefaciens EHA105 to obtain transgenic positive seedlings, 4 positive seedlings (L17, L18, L19 and L20) are selected, genome DNA is extracted, and the extraction steps are as follows:
1. placing about 100mg of fresh rice tender leaves into a 1.5ml EP tube, and grinding into powder under the condition of liquid nitrogen freezing;
2. adding 0.6ml of 2 × CTAB extracting solution (0.2% mercaptoethanol is added before use), mixing, reversing for several times, and vortexing. Water bath at 65 deg.C for 30min, and mixing by reversing every 10 min;
3. taking out the centrifuge tube, cooling, adding 0.6ml of phenol-chloroform mixed solution, mixing uniformly, suspending fully, and needing vortex;
4.12,000 rpm for 8min at room temperature;
5. transferring the supernatant (about 450. mu.L) to another new 1.5ml centrifuge tube, adding chloroform of equal volume to the supernatant, vortexing, and centrifuging at 12,000rpm for 8 min;
6. collecting supernatant of 400 μ L, adding 600 μ L anhydrous ethanol, mixing, and standing at-20 deg.C for 30 min; centrifuging at 7.4 deg.C and 12,000rpm for 20min, and discarding the supernatant;
washing the precipitate with 8.1 ml 70% ethanol (precooled), inverting for several times without vortexing, centrifuging at 7,000rpm for 3min, and discarding the supernatant;
9. washing the precipitate with 1ml 70% ethanol (precooled), inverting for several times without vortexing, centrifuging at 7,000rpm for 3min, and discarding the supernatant;
10. after ethanol is fully volatilized, 20 mu L of sterile water (containing 20 mu g/ml of RNase A) is added, and water bath is carried out at 37 ℃ for 30 min;
11. and taking 3 mu L of DNA sample to carry out agarose gel electrophoresis detection to obtain the genome DNA.
Example 2 enzymatic genomic cyclization
In order to avoid the influence of buffer in the enzyme digestion reaction system on the subsequent ligation reaction, after the components of the enzyme digestion buffer and the ligase buffer are compared, the invention adopts two enzyme digestion schemes for treatment:
1) adding a ligase buffer: adding ligase buffer during enzyme digestion, carrying out next ligation reaction without purification after the enzyme digestion is finished, and completing a ligation reaction system, wherein the enzyme digestion system is shown in table 1:
TABLE 1 enzyme digestion System
Taking 10 mu L of the enzyme-digested product, adding 1 mu L T4DNA ligase, and reacting for 5h at 16 ℃ to obtain the cyclase ligation product.
2) Adding the enzyme digestion buffer and the ligase buffer in two times: the cleavage system is shown in Table 2:
TABLE 2 enzyme digestion System
Taking 10 mu L of the enzyme-digested product, adding 1 mu L T4DNA ligase and 1 mu L of 10 Xligase buffer, and reacting for 5h at 16 ℃ to obtain the cyclase ligation product.
Example 3 specific fragment amplification
A pair of specific primers (shown as SEQ ID NO:3 and SEQ ID NO: 4) are designed on the right side of the enzyme cutting site according to the known RB terminal sequence of the T-DNA flanking sequence, the ring-formed fragments are amplified, and the fragments are reversely extended to two ends to be connected into linear fragments.
The RB terminal sequence is shown as SEQ ID NO. 2, and specifically comprises the following steps:
wherein [ aagctt ] is HindIII restriction site and [ acgt ] is TaiI restriction site
The PCR amplification system is shown in Table 3:
TABLE 3 PCR reaction amplification System
The PCR procedure was: denaturation at 95 deg.C for 5min, denaturation at 35 × (95 deg.C for 30s, 60 deg.C for 30s, and 72 deg.C for 2min), extension at 72 deg.C for 5min, and storage at 4 deg.C.
The products after the two kinds of enzyme digestion reactions are amplified, and the result is shown in figure 2 after agarose gel electrophoresis analysis, and as can be seen in the figure, the enzyme digestion buffer and the ligase buffer are added twice, the target gene bands obtained by amplification are brighter and more bands are obtained.
Recovering and purifying the amplified bands, performing one-generation singer sequencing, splicing a sequencing result to obtain a sequence between two specific primers, namely a rice genome sequence near an insertion site, comparing the sequence with a sequence in a database to obtain an insertion position, wherein the insertion position of different bands of a single sample is different and is multiple copies, and the specific result is shown in figure 3:
as can be seen from FIG. 3, a part of the same sequencing result is the vector backbone sequence (the matched part), and the other part of the sequence is compared with the rice genome sequence, so that the matched site on the genome can be found.
The sequencing results are shown in Table 4:
TABLE 4 statistical Table of sequencing results
As can be seen from the results in the table, the Tail (2) series obtained accurate localization of the transgenes of all lines on the chromosome with the best results. This is because the Tail cleavage site sequence is shorter and the compatibility is stronger.
Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein.
Sequence listing
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<120> a method for obtaining a genomic site into which a known TDNA flanking sequence is inserted
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ataatcatat attagcttat tgattctcat tttataacat tttcctttgt catatagtga 660
aattgagaca cacacaaaga aaagattttt ttttttaagt ataatacaag aacattttta 720
tttagaccat gctaacatct tcccaatttt atcagatgtc ccaaaagaac ctaatacaac 780
aacatgcatg taccaattca tatcacatag acagttaaca cgaaagttcg ttggtttttg 840
taaggaaacc aataaagcat atttgtcttc agcctctcat actatttgag aggtaaaaac 900
cgagacatta ggattattct ttatagaatg ttttggtgcc taacaaattt tactggcatt 960
tctttttttt gccataaact gttaagaggt ttatttatga attgttaact taatttcttt 1020
gatcaaacaa tagtaagttc tataaatgta tcttgtgatc tttgactatg atagacacta 1080
agctcggact catgtacata gccaaaacac tcatatatat taaaacaata atttcatcgg 1140
taaactcatg ttgtttccaa atatacaagt agagaaaacg tgtagactta ctctgaaggg 1200
acacgcatat acatactgat aaacaaccat catggagaaa tccgagccgt ttctctgcaa 1260
gccaaaccaa atttgacata tatagtaagg tcatgtgctc tatacaccta gtacctcaca 1320
ttcatttgat atctcctcta gagatacatc gatcatagtc atattttcag ctttcaatga 1380
tgatttagtc attaccattc ttgaaatata ttggtcgagt ttagtccata gatcttcgat 1440
cctgtcgtga caaagccttc catcaattgt caaaagatct ctcacgtgtg gaaatactcg 1500
atgcgtgttg ctaaatatgt gaataaaaga acgccaaatt tatagaaaaa gcgaattaaa 1560
attcaagtaa acatatgaat atttttcaag aaaagtgaat tgaaatttta ttttcatgaa 1620
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tcttatgcca ttcgaccacg atcgatagta gaggcaacta ttatggtaca gttacgagat 1740
ggacaaacaa caataacttg aaaaataatg attttttgtt ttgtttctag aaatatacac 1800
atacggatct tgaatttatc tgattgcgca actagagata tcaacattga aattcattcg 1860
cccaaatagt caatctggcg gtcacatgtc agctaacttt tccaacaatg ttcattaatt 1920
aattaacaat ctcaaagatt ttgtagattg aaatacaaat cttctctctg tggtacattt 1980
cttgaaaaat gggaaaatca agaaagtatc gaaaatgtac aaaaataaaa agaaatgaat 2040
caaagtagcc atgatcttga caacaataat cgagagagat cgtcatgata cgatttccct 2100
catccaaaat tgattttatt tcccttccca aatcaaacat atcatatgat ttcaccactc 2160
accattactt gactattctc aacaaaaaaa atattaaaaa actttatgac tttgatttta 2220
tttttatttg aagtttagcc aaaatttgaa aatatgactt ttgagaagaa aacagaataa 2280
acaaataatt agccacgcgc tatcagacag acaaaatccc acagatatgc aaagatctct 2340
cagaatcctc tccccatatc atatttttct cttttccctc tccttctttc ttcctttata 2400
aatccattta ttctcctctc atctctcagc aaaatcaaat cctcatagtt gattctctct 2460
atctctctca cgagtcacga tcctactctt cttgatcaac atgtctaatc ttcttactgt 2520
tcatcagaac ttgccagcat tgccagttga tgctacttct gatgaagttc gtaagaatct 2580
tatggatatg ttcagggacc gccaggcctt ctcagaacac acatggaaga tgcttctttc 2640
tgtttgcaga tcttgggctg cttggtgcaa gctgaacaat agaaagtggt tcccagcaga 2700
accagaagat gttcgtgatt atcttcttta tcttcaggcc cgcggcctcg ccgtcaagac 2760
aattcagcag catcttggtc aacttaatat gcttcatagg aggagcggcc tcccccgccc 2820
ttctgattca aatgctgttt ctcttgttat gaggaggatc aggaaggaga acgtggatgc 2880
tggtgaaaga gctaagcagg ccctcgcctt cgagaggaca gatttcgacc aggtgaggag 2940
cctcatggag aacagcgacc gctgccagga tattagaaat cttgcttttc ttggtattgc 3000
ttataatact cttcttcgta ttgctgaaat tgctagaatt agagttaaag atatttctag 3060
aacagatggt ggaaggatgc ttattcatat tggtagaaca aagacacttg tttctactgc 3120
tggtgttgag aaggctcttt ctcttggtgt tacaaagctg gtggagaggt ggatttctgt 3180
ttctggtgtt gctgatgatc caaataacta cctcttctgc cgcgtcagga agaatggtgt 3240
tgctgctcca tcagctactt ctcagctgtc aactagagct cttgagggca tcttcgaggc 3300
cactcatagg ctcatctacg gcgccaagga tgattcagga cagaggtacc tcgcctggag 3360
cggccacagc gcccgcgtcg gcgccgcccg cgacatggcg agggctggtg tttctattcc 3420
tgaaattatg caagctggtg gttggacaaa tgttaatatt gttatgaact acatcaggaa 3480
cttggattca gaaacaggag ctatggtgag gctgctggag gacggcgacc cgaagaagaa 3540
gaggaaggtg tagtgtatct gacgtccgta tcatcggttt cgacaacgtt cgtcaagttc 3600
aatgcatcag tttcattgcc cacacaccag aatcctacta agtttgagta ttatggcatt 3660
ggaaaagctg ttttcttcta tcatttgttc tgcttgtaat ttactgtgtt ctttcagttt 3720
ttgttttcgg acatcaaaat gcaaatggat ggataagagt taataaatga tatggtcctt 3780
ttgttcattc tcaaattatt attatctgtt gtttttactt taatgggttg aatttaagta 3840
agaaaggaac taacagtgtg atattaaggt gcaatgttag acatataaaa cagtctttca 3900
cctctctttg gttatgtctt gaattggttt gtttcttcac ttatctgtgt aatcaagttt 3960
actatgagtc tatgatcaag taattatgca atcaagttaa gtacagtata ggctttttag 4020
gtcgtagact gaattaacgc cgaattaatt cgggggatct ggattttagt actggatttt 4080
ggttttagga attagaaatt ttattgatag aagtatttta caaatacaaa tacatactaa 4140
gggtttctta tatgctcaac acatgagcga aaccctatag gaaccctaat tcccttatct 4200
gggaactact cacacattat tatggagaaa ctcgagcttg tcgatcgaca gatccggtcg 4260
gcatctactc tatttctttg ccctcggacg agtgctgggg cgtcggtttc cactatcggc 4320
gagtacttct acacagccat cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg 4380
cccgacagtc ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc 4440
atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa tgcggagcat 4500
atacgcccgg agtcgtggcg atcctgcaag ctccggatgc ctccgctcga agtagcgcgt 4560
ctgctgctcc atacaagcca accacggcct ccagaagaag atgttggcga cctcgtattg 4620
ggaatccccg aacatcgcct cgctccagtc aatgaccgct gttatgcggc cattgtccgt 4680
caggacattg ttggagccga aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc 4740
ggcccaaagc atcagctcat cgagagcctg cgcgacggac gcactgacgg tgtcgtccat 4800
cacagtttgc cagtgataca catggggatc agcaatcgcg catatgaaat cacgccatgt 4860
agtgtattga ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc 4920
ggccgcagcg atcgcatcca tagcctccgc gaccggttgt agaacagcgg gcagttcggt 4980
ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat aggtcaggct 5040
ctcgctaaac tccccaatgt caagcacttc cggaatcggg agcgcggccg atgcaaagtg 5100
ccgataaaca taacgatctt tgtagaaacc atcggcgcag ctatttaccc gcaggacata 5160
tccacgccct cctacatcga agctgaaagc acgagattct tcgccctccg agagctgcat 5220
caggtcggag acactgtcga acttttcgat cagaaacttc tcgacagacg tcgcggtgag 5280
ttcaggcttt ttcatatctc attgcccccc cggatctgcg aaagctcgag agagatagat 5340
ttgtagagag agactggtga tttcagcgtg tcctctccaa atgaaatgaa cttccttata 5400
tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt cagtggagat 5460
atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct 5520
cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc 5580
ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt 5640
gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg 5700
ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta 5760
gacgagagtg tcgtgctcca ccatgttatc acatcaatcc acttgctttg aagacgtggt 5820
tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact 5880
gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt 5940
gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc 6000
gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga 6060
gactgtatct ttgatattct tggagtagac gagagtgtcg tgtcgtagac atattcaata 6120
acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtct gaagaggagt 6180
ttagtcgtag aggagacgag tctgagactc agcgtctcgg tcgaagcttg gcactggccg 6240
tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 6300
cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 6360
aacagttgcg cagcctgaat ggcgaatgct agagcagctt gagcttggat cagattgtcg 6420
tttcccgcct tcagtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa 6480
gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag gtttatccgt 6540
tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa gtactttgat 6600
ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagg agatgatcgc 6660
ggccgggtac gtgttcgagc cgcccgcgca tgtctcaacc gtgcggctgc atgaaatcct 6720
ggccggtttg tctgatgcca agctggcggc ctggccggcc agcttggccg ctgaagaaac 6780
cgagcgccgc cgtctaaaaa ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt 6840
cgctgcgtat atgatgcgat gagtaaataa acaaatacgc aaggggaacg catgaaggtt 6900
atcgctgtac ttaaccagaa aggcgggtca ggcaagacga ccatcgcaac ccatctagcc 6960
cgcgccctgc aactcgccgg ggccgatgtt ctgttagtcg attccgatcc ccagggcagt 7020
gcccgcgatt gggcggccgt gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc 7080
ccgacgattg accgcgacgt gaaggccatc ggccggcgcg acttcgtagt gatcgacgga 7140
gcgccccagg cggcggactt ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt 7200
ccggtgcagc caagccctta cgacatatgg gccaccgccg acctggtgga gctggttaag 7260
cagcgcattg aggtcacgga tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc 7320
aaaggcacgc gcatcggcgg tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt 7380
cttgagtccc gtatcacgca gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc 7440
gttcttgaat cagaacccga gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa 7500
attaaatcaa aactcatttg agttaatgag gtaaagagaa aatgagcaaa agcacaaaca 7560
cgctaagtgc cggccgtccg agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg 7620
cagacacgcc agccatgaag cgggtcaact ttcagttgcc ggcggaggat cacaccaagc 7680
tgaagatgta cgcggtacgc caaggcaaga ccattaccga gctgctatct gaatacatcg 7740
cgcagctacc agagtaaatg agcaaatgaa taaatgagta gatgaatttt agcggctaaa 7800
ggaggcggca tggaaaatca agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt 7860
ggaggaacgg gcggttggcc aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc 7920
actggaaccc ccaagcccga ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca 7980
aatcggcgcg gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca 8040
gcggcaacgc atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg 8100
aatccgcaaa gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc 8160
caagggcgac gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga 8220
tagtcgcagc atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg 8280
cgaggtgatc cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg 8340
catggccagt gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc 8400
catgaaccga taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt 8460
tgcggacgta ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt 8520
agaaacctgc attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa 8580
gaacggccgc ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt 8640
aaagagcgaa accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg 8700
cgagatcaca gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat 8760
cgatcccggc atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga 8820
agccagatgg ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa 8880
gttctgtttc accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa 8940
ggaggaggcg gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg 9000
cgaagcatcc gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg 9060
ggaaaaaggt cgaaaagatc tctttcctgt ggatagcacg tacattggga acccaaagcc 9120
gtacattggg aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc 9180
acacatgtaa gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt 9240
aaaacttatt aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac 9300
agccgaagct cccggatacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 9360
cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac 9420
gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag 9480
agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 9540
gcgttcatcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 9600
ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 9660
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 9720
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 9780
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 9840
cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 9900
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 9960
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 10020
cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 10080
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 10140
gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 10200
agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 10260
cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 10320
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 10380
tttggtcatg cattctaggg aaggtgcgaa caagtccctg atatgagatc atgtttgtca 10440
tctggagcca tagaacaggg ttcatcatga gtcatcaact taccttcgcc gacagtgaat 10500
tcagcagtaa gcgccgtcag accagaaaag agattttctt gtcccgcatg gagcagattc 10560
tgccatggca aaacatggtg gaagtcatcg agccgtttta ccccaaggct ggtaatggcc 10620
ggcgacctta tccgctggaa accatgctac gcattcactg catgcagcat tggtacaacc 10680
tgagcgatgg cgcgatggaa gatgctctgt acgaaatcgc ctccatgcgt ctgtttgccc 10740
ggttatccct ggatagcgcc ttgccggacc gcaccaccat catgaatttc cgccacctgc 10800
tggaacagca tcaactggcc cgccaattgt tcaagaccat caatcgctgg ctggccgaag 10860
caggcgtcat gatgactcaa ggcaccttgg tcgatgccac catcattgag gcacccagct 10920
cgaccaagaa caaagagcag caacgcgatc cggagatgca tcagaccaag aaaggcaatc 10980
agtggcactt tggcatgaag gcccacattg gtgtcgatgc caagagtggc ctgacccaca 11040
gcctggtcac caccgcggcc aacgagcatg acctcaatca gctgggtaat ctgctgcatg 11100
gagaggagca atttgtctca gccgatgccg gctaccaagg ggcgccacag cgcgaggagc 11160
tggccgaggt ggatgtggac tggctgatcg ccgagcgccc cggcaaggta agaaccttga 11220
aacagcatcc acgcaagaac aaaacggcca tcaacatcga atacatgaaa gccagcatcc 11280
gggccagggt ggagcaccca tttcgcatca tcaagcgaca gttcggcttc gtgaaagcca 11340
gatacaaggg gttgctgaaa aacgataacc aactggcgat gttattcacg ctggccaacc 11400
tgtttcgggc ggaccaaatg atacgtcagt gggagagatc tcactaaaaa ctggggataa 11460
cgccttaaat ggcgaagaaa cggtctaaat aggctgattc aaggcattta cgggagaaaa 11520
aatcggctca aacatgaaga aatgaaatga ctgagtcagc cgagaagaat ttccccgctt 11580
attcgcacct tccctaggta ctaaaacaat tcatccagta aaatataata ttttattttc 11640
tcccaatcag gcttgatccc cagtaagtca aaaaatagct cgacatactg ttcttccccg 11700
atatcctccc tgatcgaccg gacgcagaag gcaatgtcat accacttgtc cgccctgccg 11760
cttctcccaa gatcaataaa gccacttact ttgccatctt tcacaaagat gttgctgtct 11820
cccaggtcgc cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt taaaaaatca 11880
tacagctcgc gcggatcttt aaatggagtg tcctcttccc agttttcgca atccacatcg 11940
gccagatcgt tattcagtaa gtaatccaat tcggctaagc ggctgtctaa gctattcgta 12000
tagggacaat ccgatatgtc gatggagtga aagagcctga tgcactccgc atacagctcg 12060
ataatctttt cagggctttg ttcatcttca tactcttccg agcaaaggac gccatcggcc 12120
tcactcatga gcagattgct ccagccatca tgccgttcaa agtgcaggac ctttggaaca 12180
ggcagctttc cttccagcca tagcatcatg tccttttccc gttccacatc ataggtggtc 12240
cctttatacc ggctgtccgt catttttaaa tataggtttt cattttctcc caccagctta 12300
tataccttag caggagacat tccttccgta tcttttacgc agcggtattt ttcgatcagt 12360
tttttcaatt ccggtgatat tctcatttta gccatttatt atttccttcc tcttttctac 12420
agtatttaaa gataccccaa gaagctaatt ataacaagac gaactccaat tcactgttcc 12480
ttgcattcta aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg 12540
cgtataacat agtatcgacg gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc 12600
tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg 12660
cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct gccgccttac 12720
aacggctctc ccgctgacgc cgt 12743
<210> 2
<211> 322
<212> DNA
<213> vector sequence (TDNA sequence)
<400> 2
gtgtcgtgct ccaccatgtt ggcaagctgc tctagggaag agcagtctga gactcaggct 60
cttcggtcga agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 120
cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 180
agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgctagag 240
cagcttgagc ttggatcaga ttgtcgtttc ccgccttcag tttaaactat cagtgtttga 300
caggatatat tggcgggtaa ac 322
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caaggcgatt aagttgggta acgc 24
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gccttgcagc acatccccct ttcg 24
Claims (8)
1. A method for obtaining a genomic site for insertion of a flanking sequence of a known TDNA, comprising the steps of:
s1, transforming a vector containing a known TDNA flanking sequence into a plant by an agrobacterium-mediated method to obtain a transgenic positive seedling;
s2, finding a 4-base restriction enzyme cutting site and/or a 6-base restriction enzyme cutting site at the RB end of the known TDNA flanking sequence;
s3, extracting genome DNA of the transgenic positive seedling, and selecting restriction enzymes with 4-base enzyme cutting sites and/or 6-base enzyme cutting sites to perform enzyme cutting operation on the genome DNA to obtain gene fragments with different lengths;
s4, carrying out enzyme linking operation on the gene fragment by using DNA ligase to obtain a ring forming fragment; according to the right sequence of the enzyme cutting site, a pair of specific primers is designed to amplify the cyclized segment, and the fragments are reversely extended to two ends and connected into linear segments;
and S5, recovering and purifying the linear fragment, sending the linear fragment to a first generation sequencing, and comparing the obtained sequencing result with the genome data in a database to obtain the information of the genomic locus where the known TDNA flanking sequence is inserted.
2. The method of claim 1, wherein the RB end sequence of the known TDNA flanking sequence is shown in SEQ ID NO 2 in step S2.
3. The method for obtaining the genomic site with the inserted flanking sequence of the known TDNA of claim 1, wherein the 4-base cleavage site is Tail cleavage site in step S2.
4. The method of claim 1, wherein the 6-base cleavage site is Hind iii cleavage site in step S2.
5. The method for obtaining the genomic site with the inserted flanking sequence of known TDNA according to claim 1, wherein the specific digestion reaction process of the Tail cleavage site in step S3 is as follows: mu.L of genomic DNA, 1. mu.L of 10 Xdigestion buffer and 1. mu.L of Tail endonuclease were added, and the mixture was reacted at 65 ℃ for 4 hours, followed by heat inactivation at 80 ℃.
6. The method of claim 1, wherein in step S3, the Hind iii site is cut by the following steps: mu.L of genomic DNA, 1. mu.L of 10 Xdigestion buffer, and 1. mu.L of HindIII endonuclease were added, and the mixture was reacted at 37 ℃ for 4 hours, followed by heat inactivation at 80 ℃.
7. The method of claim 1, wherein in step S4, the enzymatic ligation reaction is performed as follows: mu.L of the digested product, 1. mu. L T4 ligase and 1. mu.L of 10 Xligase buffer were added, and the mixture was reacted at 16 ℃ for 5 hours, followed by storage at 4 ℃.
8. The method of claim 1, wherein the specific primer sequences shown in SEQ ID NO 3 and SEQ ID NO 4 are used in step S4 to obtain the known TDNA flanking sequence inserted into the genomic locus.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7220893B1 (en) * | 1999-05-19 | 2007-05-22 | Cornell Research Foundation, Inc. | Plasmids and methods for construction of non-redundant, indexed, saturation, gene-disruption plant and animal libraries |
| CN107190003A (en) * | 2017-06-09 | 2017-09-22 | 武汉天问生物科技有限公司 | A kind of method of efficient quick separating T DNA insertion point flanking sequences and application thereof |
| CN112359038A (en) * | 2020-11-07 | 2021-02-12 | 浙江大学 | Cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanking sequence and application thereof |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7220893B1 (en) * | 1999-05-19 | 2007-05-22 | Cornell Research Foundation, Inc. | Plasmids and methods for construction of non-redundant, indexed, saturation, gene-disruption plant and animal libraries |
| CN107190003A (en) * | 2017-06-09 | 2017-09-22 | 武汉天问生物科技有限公司 | A kind of method of efficient quick separating T DNA insertion point flanking sequences and application thereof |
| CN112359038A (en) * | 2020-11-07 | 2021-02-12 | 浙江大学 | Cyst nematode resistant transgenic soybean event ZHs1-2 exogenous insert flanking sequence and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 化工仪器网: "反向PCR （inverse-PCR）实验步骤", Retrieved from the Internet <URL:https://www.chem17.com/tech_news/detail/2244495.html> * |
| 道客巴巴: "T-DNA***侧翼序列的分离方法", Retrieved from the Internet <URL:https://www.doc88.com/p-9932381112263.html> * |
| 马硕;焦悦;杨江涛;王旭静;王志兴;: "基因组测序技术解析耐除草剂转基因水稻G2-7的分子特征", 作物学报, no. 11 * |
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