CN107630079A - The method for determining the sequence of exogenous dna fragment, insertion position and marginal sequence in genetically modified organism - Google Patents
The method for determining the sequence of exogenous dna fragment, insertion position and marginal sequence in genetically modified organism Download PDFInfo
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Abstract
The invention discloses the method for determining the sequence of exogenous dna fragment, insertion position and marginal sequence in genetically modified organism.Method disclosed by the invention includes：The full-length genome of genetically modified organism is sequenced, sequence A and genetically modified organism whole genome sequence are subjected to sequence alignment, determine the sequence of exogenous dna fragment in genetically modified organism, insertion position and marginal sequence；Genetically modified organism is the genetically modified organism obtained using the vehicle treated receptor biological containing exogenous dna fragment；Sequence A is a1), a2) or a3)：A1) the full length sequence of carrier；A2) the sequence of any DNA fragmentation containing exogenous dna fragment from carrier；The sequence of a3 exogenous dna fragments or any of which fragment.The method of the present invention can break through species limitation, not limited by target gene used when building genetically modified organism, can more detect transgenosis insertion position, marginal sequence even copy number fast accurate.
Description
Technical field
The present invention relates to determined in biological technical field the sequence of exogenous dna fragment in genetically modified organism, insertion position and
The method of marginal sequence.
Background technology
In recent years, the safety evaluation on transgenic organism particularly GM food, social pass is increasingly becomed
The focal issue of note.Insetion sequence, insertion position and its marginal sequence of the transgenosis in acceptor gene group are specified, is to assess to divide
Analyse the key link of transgenosis safe.Around this problem, people can use the clear and definite transgenosis of Southern hybridizing methods
Copy number, with gradually obtaining insertion position and its marginal sequence of the transgenosis in acceptor gene group the methods of chromosome walking.
But these methods, many times can by transgene copy number, homologous gene similitude, transgenic sequence, genome sequence,
The limitation of the factors such as T-DNA areas uncertainty, it is generally inefficient.Need to develop a kind of fast accurate for this and efficiently determine
The method of the sequence of exogenous dna fragment and/or insertion position and/or marginal sequence in genetically modified organism.
The content of the invention
The technical problems to be solved by the invention be how to determine in genetically modified organism the sequence of exogenous dna fragment and/or
Insertion position and/or marginal sequence.
In order to solve the above technical problems, present invention firstly provides the sequence for determining exogenous dna fragment in genetically modified organism
And/or the method for insertion position and/or marginal sequence.
The sequence of exogenous dna fragment and/or insertion position and/or limit in determination genetically modified organism provided by the present invention
The method of sequence, including it is following 1) and 2)：
1) full-length genome of genetically modified organism is sequenced, obtains genetically modified organism whole genome sequence；
2) sequence A and the genetically modified organism whole genome sequence are subjected to sequence alignment, determine the genetically modified organism
The sequence of middle exogenous dna fragment and/or insertion position and/or marginal sequence；The genetically modified organism is using containing described outer
The genetically modified organism that the vehicle treated receptor biological of source DNA fragment obtains；
The sequence A is a1), a2) or a3)：
A1) the full length sequence of the carrier；
A2) the sequence of any DNA fragmentation containing the exogenous dna fragment from the carrier；
A3) the sequence of the exogenous dna fragment or any of which fragment.
In the above method, step 2) may include it is following 21) and 22)：
21) the sequence A and the genetically modified organism whole genome sequence are subjected to sequence alignment, obtain the transgenosis
The sequence matched in biological whole genome sequence with the sequence A, it is sequence B by the sequence designations；
22) reference sequences of the sequence B and the receptor biological are subjected to sequence alignment, determine the transgenosis life
The sequence of exogenous dna fragment and/or insertion position and/or marginal sequence in thing.
In the above method, step 22) may include following 22a) and 22b)：
The reference sequences of the sequence B and the receptor biological 22a) are subjected to sequence alignment, primarily determines that and described turns base
Because of the sequence of exogenous dna fragment and/or insertion position in biology and/or marginal sequence；
Primer 22b) is designed according to the reference sequences of known array in the sequence B and the receptor biological, using described
The genome of genetically modified organism described in primer pair is expanded, and further determines that the transgenosis is given birth to according to the sequence of amplified production
The sequence of exogenous dna fragment and/or insertion position and/or marginal sequence in thing.
Step 22b) in, the amplification can be that PCR is expanded.The sequence of the amplified production can be by by the amplified production
Generation sequencing is carried out to obtain.
Because sequencing overburden depth is limited, unknown nucleotide sequence is there may be in the genetically modified organism whole genome sequence, institute
Also likely to be present unknown nucleotide sequence in the sequence B, or the sequence B can not be covered all described in the genetically modified organism
The marginal sequence of the insertion position of exogenous dna fragment and the exogenous dna fragment.Step 22b) it is middle using described in the primer pair
It is in order to by institute in the unknown nucleotide sequence in the sequence B and the genetically modified organism that the genome of genetically modified organism, which carries out amplification,
The marginal sequence amplification for stating the insertion position of the unlapped exogenous dna fragment of sequence B and the exogenous dna fragment comes out,
And know in the sequence B that sequence B described in unknown nucleotide sequence and the genetically modified organism is unlapped described outer by sequencing
The marginal sequence of the insertion position of source DNA fragment and the exogenous dna fragment.
Step 22b) in, the sequence according to amplified production further determines that exogenous DNA piece in the genetically modified organism
The sequence and/or insertion position and/or marginal sequence of section can be by by the sequence of the amplified production and the receptor biologicals
Reference sequences carry out sequence alignment to realize.
In the above method, the reference sequences of the receptor biological are the full-length genome sequence of the wild type of the receptor biological
Row.
In the above method, the full-length genome to genetically modified organism is carried out sequencing and carried out using high-flux sequence platform.
The high-flux sequence platform concretely HiSEQ2500 microarray datasets.
In the above method, methods described may additionally include the full-length genome of genetically modified organism is sequenced before to described turn
Library used in the genome structure sequencing of gene biological.
Library used in the sequencing can be the library that clip size is 500bp-2Kb, such as the library that clip size is 1Kb.
In the above method, using high-flux sequence platform to the genome sequencing of genetically modified organism after, sequence can be passed through
Splicing software and/or module carry out sequence assembly and obtain the genetically modified organism whole genome sequence.
The sequence assembly software can be SOAPdenovo2 softwares, such as SOAPdenovo2 (Version 2.04:
Released on July 13th, 2012) software.
Methods described may additionally include using high-flux sequence platform to the genome sequencing of genetically modified organism after, remove
Low quality data, invalid data and/or joint contamination data, obtain clean data in sequencing result.The sequence assembly can be
Sequence assembly is carried out to the clean data.
In the above method, the sequence B can be overlap (contig) or Frame sequence (scaffold).It is described overlapping
Unknown nucleotide sequence is not present to carry out the known sequence of sequence obtained after sequence assembly in sequence that is, in sequence.The framework sequence
It is classified as after carrying out sequence assembly and the unknown sequence of partial nucleotide (base) is also present.
In the above method, the genetically modified organism can be genetically modified plants or transgenic animals.The receptor biological can be
Plant or animal.
Above, sequence alignment can use sequence alignment program and/or module to carry out.The sequence alignment program can be
Clustal softwares, such as ClustalW.
In order to solve the above technical problems, present invention also offers determine exogenous dna fragment copy number in genetically modified organism
Method.
The method of exogenous dna fragment copy number includes in determination genetically modified organism provided by the present invention：By using institute
State and determine that the sequence of exogenous dna fragment and/or the method for insertion position and/or marginal sequence determine transgenosis in genetically modified organism
The sequence of exogenous dna fragment and/or insertion position and/or marginal sequence are outer described in the genetically modified organism to determine in biology
The copy number of source DNA fragment.
In the above method, the genetically modified organism can be genetically modified plants or transgenic animals.
In order to solve the above technical problems, present invention also offers determine genetically modified organism in exogenous dna fragment sequence and/
Or the system of insertion position and/or marginal sequence.
The sequence of exogenous dna fragment and/or insertion position and/or limit in determination genetically modified organism provided by the present invention
The system of sequence includes following b1)-b4) at least two：
B1 the reagent and/or instrument needed for high-flux sequence) are carried out；
B2) carry out expanding required reagent and/or instrument；
B3) sequence alignment program and/or module；
B4) sequence assembly software and/or module.
Said system also can be only by above-mentioned b1)-b4) at least two form.
In said system, the genetically modified organism can be genetically modified plants or transgenic animals.
In order to solve the above technical problems, present invention also offers following any applications：
P1) the sequence of exogenous dna fragment in genetically modified organism and/or insertion position and/or the marginal sequence of determining
The application of method or the method for determining exogenous dna fragment copy number in genetically modified organism in Biology Breeding；
P2) the sequence of exogenous dna fragment in genetically modified organism and/or insertion position and/or the marginal sequence of determining
Whether method or the method for determining exogenous dna fragment copy number in genetically modified organism are in evaluation genetically modified organism in safety
Using；
P3) system is it is determined that the sequence of exogenous dna fragment and/or insertion position and/or limit in genetically modified organism
Application in sequence；
P4) system is it is determined that application in genetically modified organism in exogenous dna fragment copy number；
P5) application of the system in Biology Breeding；
P6) system evaluation genetically modified organism whether the application in safety.
In above-mentioned application, the genetically modified organism can be genetically modified plants or transgenic animals.The biology can be plant
Or animal.
The method of the present invention has the following advantages that：
1) species limitation can be broken through, the sequence of carrier used is either just known that in known structure genetically modified organism
, can be by the true genetically modified organism of the bright method of this method when building the sequence of target gene used during genetically modified organism
Insetion sequence, insertion position and the marginal sequence for the exogenous dna fragment being actually inserted into；
2) target gene used is limited when can not be by structure genetically modified organism, is more widely detected transgenosis and is carried
The transgenosis of non-targeted gene is got a lift effect in body, can determine the sequence being inserted into carrier in receptor biological genome simultaneously；
3) foreign gene insertion position, marginal sequence even copy number can more be detected fast accurate；
4), may in genetically modified organism whole genome sequence when carrying out high-flux sequence because sequencing overburden depth is limited
Unknown nucleotide sequence be present, method of the invention can be outer in the case where that need not learn the complete genome sequence of genetically modified organism
Insetion sequence, insertion position and the marginal sequence of source DNA fragment.
Brief description of the drawings
Fig. 1 is target gene AtD-CGS expression cassettes sequence and scaffold99334 comparison result in transgene carrier.
Fig. 2 be with ClustalW softwares to target gene AtD-CGS expression cassettes sequence in transgene carrier with
Scaffold99334 (before unknown base of plugging a gap) and scaffold99334.fill (after unknown base of plugging a gap) is carried out
The result of comparison.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The method of the sequence of exogenous dna fragment, insertion position and marginal sequence in the determination genetically modified organism of the present invention, bag
Include following 1) -5)：
1) full-length genome of genetically modified organism is sequenced using high-flux sequence platform, obtains the full base of genetically modified organism
Because of a group sequence；
2) sequence A and genetically modified organism whole genome sequence are subjected to sequence alignment, determine exogenous DNA in genetically modified organism
Sequence, insertion position and the marginal sequence of fragment；
Sequence A is a1), a2) or a3)：
A1 the full length sequence for the carrier used during the genetically modified organism) is prepared；
A2) in carrier any DNA fragmentation containing exogenous dna fragment sequence；
A3) the sequence of exogenous dna fragment or any of which fragment.
3) sequence A and genetically modified organism whole genome sequence are subjected to sequence alignment, obtain genetically modified organism full-length genome
The sequence matched in sequence with sequence A, it is sequence B by the sequence designations；
4) sequence B and the wild-type biology whole genome sequence as reference sequences are subjected to sequence alignment, primarily determined that
The sequence of exogenous dna fragment, insertion position and marginal sequence in genetically modified organism；
5) primer is designed according to known array in sequence B and reference sequences, utilizes the gene of the primer pair genetically modified organism
Group is expanded, and the sequence of exogenous dna fragment, insertion position in genetically modified organism are further determined that according to the sequence of amplified production
With marginal sequence.
It is specifically described below by taking genetically engineered soybean as an example and determines the sequence of exogenous dna fragment in genetically modified organism, insertion position
Put the method with marginal sequence.
Embodiment 1, turn the sequence of exogenous dna fragment in AtDCGS homomethionin soybean, insertion position and marginal sequence
It is determined that
First, AtDCGS homomethionin soybean CGS-ZG11 preparation is turned
Using carrier pGPTV-Bar-DCGS, by agriculture bacillus mediated transgenic method, AtD-CGS (is also known as
AtDCGS) genetic transformation is to soybean varieties Zi Gong winter beans, obtains turning AtD-CGS homomethionin soybean CGS-ZG11 (turning AtD-
CGS homomethionin genetically engineered soybeans strain (Han Qingmei etc., the identification of homomethionin genetically engineered soybean and genetic stability analysis,
Chinese oil crops journal, 2015,37 (6):789-796)).
2nd, sequence, insertion position and the marginal sequence of exogenous dna fragment in AtD-CGS homomethionin soybean CGS-ZG11 are turned
The determination of row
1st, the preparation of AtD-CGS homomethionin soybean CGS-ZG11 genomic DNA sample is turned
AtD-CGS is turned using the plant genome DNA extracts kit extraction of TIANGEN Biotech (Beijing) Co., Ltd.
Homomethionin soybean CGS-ZG11 genomic DNA.
2nd, the genome for turning AtD-CGS homomethionin soybean resurveys sequence
Based on HiSEQ2500 sequencing technologies platforms, using double end sequencings (Paired-End) method, structure genome is surveyed
The small fragment library of sequence, obtain raw sequencing data.And then joint pollution and low quality data are removed, obtain clean data
(clean data), the standard for removing data is as follows：A. when can not determine that the ratio of base information is more than in single-ended sequencing read
, it is necessary to remove this to reads when 10%；B. when the low quality i.e. Phred values contained in single-ended sequencing read are less than 5 base number
More than this read length ratios 50% when, it is necessary to remove this to reads.Turn the genome weight of ATCGS homomethionin soybean
Cleaning sequence in sequencing data and the sequence containing indefinite base N are as shown in table 1.
Table 1, the genome weight sequencing data for turning AtDCGS homomethionin soybean
| Library | Clean sequence (Clean Reads) | The ratio of high quality Q30 sequences |
| 500bp | 159,451,342 | 89.57% |
1) SOAPdenovo2 (Version 2.04 are used:Released on July 13th, 2012) software combination base
Because group, relevant information are as shown in table 2.
Table 2, turn AtDCGS homomethionin soybean genome assembling result
| Soybean reference gene group size | 978495272 bp |
| Genome assembling size (containing N) | 964714736 bp |
| Genome assembling size (being free of N) | 885873140 bp |
| Scaffold numbers | 1249045 |
| Scaffold average lengths | 772 bp |
| Scaffold median lengths | 127 bp |
| Scaffold extreme lengths | 422777 bp |
| Scaffold shortest lengths | 100 bp |
| N50 Scaffold length | 20840 bp |
| Contig numbers | 1985644 |
| Contig average lengths | 469 bp |
| Contig median lengths | 137 bp |
| Contig extreme lengths | 51715 bp |
| Contig shortest lengths | 100 bp |
| N50 Contig length | 2783 bp |
In table 2, soybean reference gene group is Williams 82 whole genome sequence, the soybean whole genome sequence
Version number is Gmax_275_Wm82.a2.v1.
2) according to target gene AtD-CGS expression cassettes sequence (seed specific promoters in pGPTV-Bar-DCGS
(LegB4) --- lead peptide (TP) --- target gene (AtD-CGS) --- terminator (TOCT)), compare analysis Scaffold sequences
Row, it is found that target gene AtD-CGS expression cassettes sequence-specific is compared onto scaffold99334 (55746bp), such as Fig. 1 (figures
Scaffo represents that scaffold99334, DCGS- represent target gene AtD-CGS expression cassettes sequence in 1) shown in, and
It is located at scaffold99334 sequences with the sequence in target gene AtDCGS expression cassette sequences match in scaffold99334
Center section.
3) by the scaffold99334 sequence alignments of acquisition to the soybean reference gene group in table 2, find
Scaffold99334 is located at No. 09 chromosome Chr09:Between 39880882-39924565, and with ClustalW softwares pair
The scaffold99334 and reference sequences section (No. 09 chromosome Chr09:It is careful to be carried out between 39880882-39924565)
Compare, it is found that the exogenous sequences of underscore mark are inserted into Chr09:Between 39917686-39917762, as shown in Figure 2.
4) due to overburden depth problem is sequenced, exogenous sequences insetion sequence and insertion position limit in scaffold99334
Sequence is present at unknown nucleotide sequence one (Fig. 2).According to this unknown nucleotide sequence both sides sequence, design primer CG-F and CG-R (such as table 3) expand
Increase unknown nucleotide sequence, and unknown nucleotide sequence is subjected to generation sequencing.
Table 3, the primer for expanding unknown nucleotide sequence
| Primer | Sequence |
| CG-F | 5'-AGGGCTGCTAAAGGAAGCGGAACA-3' |
| CG-R | 5'-CGATGTAGTGGTTGACGATGGTG-3' |
| GT-F | 5'-GCCTGAAAATGAGGAAGAAACA-3' |
| GT-R | 5'-CAATGAATCAACAACTCTCCTGGCG-3' |
| GA-F | 5'-ACACTCAACCCTATCTCGGGCTATT-3' |
| GA-R | 5'-GGTATCTTATGGCTGCTTGGAGTTG-3' |
5) sequence alignment for unknown nucleotide sequence being sequenced to obtain clearly turns ATCGS height into the soybean reference gene group in table 2
Exogenous sequences insertion position and marginal sequence in methionine soybean.As shown in Fig. 2 exogenous sequences (italic) are inserted into soybean the 09th
Number chromosome Chr09:Between 39917686-39917762, the side at insertion point both ends can be learnt according to soybean reference sequences
Border sequence.
6 simultaneously clear and definite exogenous sequences details.As shown in Fig. 2 the exogenous sequences of insertion reach 9343bp, such as sequence table
Shown in middle sequence 1, in sequence 1,171-423 positions are NOS terminator sequence, and 494-2305 positions are gus gene sequence, the
2350-3061 positions are TOCT terminator sequences, and 3132-4535 positions are AtD-CGS gene orders, and 4719-7472 positions are
LegB4 promoter sequences, 7513-8125 positions are pNOS promoter sequences, and 8126-8714 is herbicide resistance gene Bar
Gene order, 8715-8937 positions are the terminator sequences of transcript 7.This exogenous sequences information and destination carrier one
Cause.Insertion position information is utilized simultaneously, respectively in upstream and downstream joint, designs two couples of primers (GT-F/GT-R and GA-F/GA-R
(i.e. GT-F/GT-R and GA-F/GA-R in table 3)), enter performing PCR amplification, by conventional sequencing, confirm insertion position, show, profit
Determine to turn with the method for the sequence of exogenous dna fragment, insertion position and marginal sequence in the determination genetically modified organism of the present invention
The result of the sequence of exogenous dna fragment, insertion position and marginal sequence is reliable in AtDCGS homomethionin soybean.
6) according to target gene AtD-CGS expression cassettes sequence (seed specific promoters in pGPTV-Bar-DCGS
(LegB4) --- lead peptide (TP) --- target gene (AtD-CGS) --- terminator (TOCT)), compare analysis Scaffold sequences
Row, find in addition to scaffold99334, are not detected by other sequences that can be specifically compared with target gene expression cassette, explanation
Target gene is that single copy is present in the genome for turning AtD-CGS homomethionin soybean CGS-ZG11, this to it is related
Southern results of hybridization (make by Han Qingmei etc., the identification of homomethionin genetically engineered soybean and genetic stability analysis, Chinese oil plant
Thing journal, 2015,37 (6):It is 789-796) consistent.Illustrate that this method also has the advantages of can detecting copy number.
Claims (10)
1. the sequence and/or the method for insertion position and/or marginal sequence of exogenous dna fragment in genetically modified organism are determined, including
It is following 1) and 2)：
1) full-length genome of genetically modified organism is sequenced, obtains genetically modified organism whole genome sequence；
2) sequence A and the genetically modified organism whole genome sequence are subjected to sequence alignment, determine the genetically modified organism China and foreign countries
The sequence of source DNA fragment and/or insertion position and/or marginal sequence；The genetically modified organism is to utilize to contain the exogenous DNA
The genetically modified organism that the vehicle treated receptor biological of fragment obtains；
The sequence A is a1), a2) or a3)：
A1) the full length sequence of the carrier；
A2) the sequence of any DNA fragmentation containing the exogenous dna fragment from the carrier；
A3) the sequence of the exogenous dna fragment or any of which fragment.
2. according to the method for claim 1, it is characterised in that：Step 2) include it is following 21) and 22)：
21) the sequence A and the genetically modified organism whole genome sequence are subjected to sequence alignment, obtain the genetically modified organism
The sequence matched in whole genome sequence with the sequence A, it is sequence B by the sequence designations；
22) reference sequences of the sequence B and the receptor biological are subjected to sequence alignment, determine the genetically modified organism China and foreign countries
The sequence of source DNA fragment and/or insertion position and/or marginal sequence.
3. according to the method for claim 2, it is characterised in that：Step 22) includes following 22a) and 22b)：
The reference sequences of the sequence B and the receptor biological 22a) are subjected to sequence alignment, primarily determine that the transgenosis life
The sequence of exogenous dna fragment and/or insertion position and/or marginal sequence in thing；
Primer 22b) is designed according to the reference sequences of known array in the sequence B and the receptor biological, utilizes the primer
The genome of the genetically modified organism is expanded, further determined that according to the sequence of amplified production in the genetically modified organism
The sequence of exogenous dna fragment and/or insertion position and/or marginal sequence.
4. according to any described method in claim 1-3, it is characterised in that：The full-length genome to genetically modified organism enters
Row sequencing is carried out using high-flux sequence platform.
5. according to any described method in claim 1-4, it is characterised in that：Methods described is additionally included in genetically modified organism
Full-length genome be sequenced before to library used in the structure sequencing of the genome of the genetically modified organism.
6. according to any described method in claim 1-5, it is characterised in that：Transgenosis is given birth to using high-flux sequence platform
After the genome sequencing of thing, it is complete that the genetically modified organism is obtained by sequence assembly software and/or module progress sequence assembly
Genome sequence.
7. according to any described method in claim 1-6, it is characterised in that：The genetically modified organism be genetically modified plants or
Transgenic animals.
8. the method for exogenous dna fragment copy number in genetically modified organism is determined, including：By using any in claim 1-8
Methods described determines in genetically modified organism the sequence of exogenous dna fragment and/or insertion position and/or marginal sequence come described in determining
The copy number of exogenous dna fragment described in genetically modified organism.
9. the sequence and/or the system of insertion position and/or marginal sequence of exogenous dna fragment in genetically modified organism are determined, including
Following b1)-b4) at least two：
B1 the reagent and/or instrument needed for high-flux sequence) are carried out；
B2) carry out expanding required reagent and/or instrument；
B3) sequence alignment program and/or module；
B4) sequence assembly software and/or module.
10. following any applications：
P1) application of any methods described in Biology Breeding in claim 1-8；
P2) in claim 1-8 any methods described evaluation genetically modified organism whether the application in safety；
P3) system described in claim 9 it is determined that in genetically modified organism the sequence of exogenous dna fragment and/or insertion position and/or
Application in marginal sequence；
P4) system described in claim 9 is it is determined that application in genetically modified organism in exogenous dna fragment copy number；
P5) application of the system described in claim 9 in Biology Breeding；
P6) system described in claim 9 evaluation genetically modified organism whether the application in safety.
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| CN110600079B (en) * | 2019-08-12 | 2021-12-10 | 中国水稻研究所 | Transgene identification method and identification device |
| CN110556165A (en) * | 2019-09-12 | 2019-12-10 | 浙江大学 | method for rapidly identifying transgene or gene editing material and insertion site thereof by using whole genome re-sequencing data |
| WO2021047363A1 (en) * | 2019-09-12 | 2021-03-18 | 浙江大学 | Method for using whole genome re-sequencing data to quickly identify transgenic or gene editing material and insertion sites thereof |
| CN110556165B (en) * | 2019-09-12 | 2022-03-18 | 浙江大学 | Method for rapidly identifying transgene or gene editing material and insertion site thereof |
| EP3919629A4 (en) * | 2019-09-12 | 2022-06-15 | Zhejiang University | Method for using whole genome re-sequencing data to quickly identify transgenic or gene editing material and insertion sites thereof |
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