CN107630079B - Method for determining the sequence, insertion position and border sequence of foreign DNA fragments in transgenic organisms - Google Patents

Abstract

The invention discloses a method for determining the sequence, insertion position and border sequence of exogenous DNA fragment in transgenic organism. The method disclosed by the invention comprises the following steps: sequencing the whole genome of the transgenic organism, and comparing the sequence A with the whole genome sequence of the transgenic organism to determine the sequence, the insertion position and the marginal sequence of the exogenous DNA fragment in the transgenic organism; the transgenic organism is obtained by processing a receptor organism by using a vector containing an exogenous DNA fragment; sequence a is a1), a2) or a 3): a1) the full-length sequence of the vector; a2) the sequence of any DNA fragment containing the exogenous DNA fragment from the vector; a3 exogenous DNA fragment or the sequence of any one of the fragments. The method of the invention can break through species limitation, is not limited by target genes used in constructing transgenic organisms, and can detect the position of the transgene insertion, the marginal sequence and even the copy number more quickly and accurately.

Description

Determination of sequences, insertion positions and marginal sequences of foreign DNA fragments in GMOs Methods

technical field

The present invention relates to a method for determining the sequence, insertion position and marginal sequence of exogenous DNA fragments in transgenic organisms in the field of biotechnology.

Background technique

In recent years, the safety assessment of genetically modified organisms, especially genetically modified foods, has increasingly become the focus of social concern. Identifying the insertion sequence, insertion position and marginal sequence of the transgene in the recipient genome is the key link in evaluating and analyzing the safety of the transgene. Around this problem, people can use Southern hybridization method to determine the copy number of the transgene, and use methods such as chromosome walking to gradually obtain the insertion position of the transgene in the recipient genome and its marginal sequence. However, these methods are often limited by factors such as transgene copy number, homologous gene similarity, transgene sequence, genome sequence, T-DNA region uncertainty and other factors, and the overall efficiency is not high. To this end, it is necessary to develop a fast, accurate and efficient method for determining the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in transgenic organisms.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to determine the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in transgenic organisms.

In order to solve the above technical problems, the present invention first provides a method for determining the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in transgenic organisms.

The method for determining the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in transgenic organisms provided by the present invention includes the following 1) and 2):

1) Sequencing the whole genome of the transgenic organism to obtain the whole genome sequence of the transgenic organism;

2) Sequence comparison of sequence A with the whole genome sequence of the transgenic organism to determine the sequence and/or insertion position and/or marginal sequence of the exogenous DNA fragment in the transgenic organism; The transgenic organism obtained by processing the recipient organism with the vector of the source DNA fragment;

Said sequence A is a1), a2) or a3):

a1) the full-length sequence of the vector;

a2) the sequence of any DNA fragment from the vector containing the exogenous DNA fragment;

a3) The sequence of the exogenous DNA fragment or any fragment thereof.

In the above method, step 2) can include the following 21) and 22):

21) aligning the sequence A with the complete genome sequence of the transgenic organism to obtain a sequence matching the sequence A in the complete genome sequence of the transgenic organism, and naming the sequence as sequence B;

22) Sequence alignment of the sequence B with the reference sequence of the recipient organism to determine the sequence and/or insertion position and/or marginal sequence of the exogenous DNA fragment in the transgenic organism.

In the above method, step 22) can include the following 22a) and 22b):

22a) aligning the sequence B with the reference sequence of the recipient organism, and preliminarily determining the sequence and/or the insertion position and/or the marginal sequence of the exogenous DNA fragment in the transgenic organism;

22b) Design primers according to the known sequence in the sequence B and the reference sequence of the recipient organism, use the primers to amplify the genome of the transgenic organism, and further determine the transgenic organism according to the sequence of the amplified product The sequence and/or insertion position and/or marginal sequence of the exogenous DNA fragment in .

In step 22b), the amplification may be PCR amplification. The sequence of the amplification product can be obtained by first-generation sequencing of the amplification product.

Due to the limited depth of sequencing coverage, unknown sequences may exist in the whole genome sequence of the transgenic organism, so there may also be unknown sequences in the sequence B, or the sequence B cannot fully cover the exogenous DNA fragments in the transgenic organism and the marginal sequence of the insertion position of the exogenous DNA fragment. In step 22b), the use of the primers to amplify the genome of the transgenic organism is to amplify the unknown sequence in the sequence B and the exogenous DNA fragments and all the sequences not covered by the sequence B in the transgenic organism. The marginal sequence of the insertion position of the exogenous DNA fragment is amplified, and the unknown sequence in the sequence B and the exogenous DNA fragment and the exogenous DNA not covered by the sequence B in the transgenic organism are obtained by sequencing. The marginal sequence of the insertion position of the DNA fragment.

In step 22b), the further determination of the sequence and/or the insertion position and/or the marginal sequence of the exogenous DNA fragment in the transgenic organism according to the sequence of the amplification product can be performed by comparing the sequence of the amplification product with the receptor. Alignment of biological reference sequences.

In the above method, the reference sequence of the recipient organism is the whole genome sequence of the wild-type of the recipient organism.

In the above method, the whole genome of the transgenic organism is sequenced using a high-throughput sequencing platform. The high-throughput sequencing platform may specifically be a HiSEQ2500 sequencing platform.

In the above method, the method may further comprise constructing a library for sequencing the genome of the transgenic organism before sequencing the whole genome of the transgenic organism.

The library used for the sequencing can be a library with a fragment size of 500bp-2Kb, such as a library with a fragment size of 1Kb.

In the above method, after the high-throughput sequencing platform is used to sequence the whole genome of the transgenic organism, the whole genome sequence of the transgenic organism can be obtained by performing sequence splicing through sequence splicing software and/or modules.

The sequence splicing software may be SOAPdenovo2 software, such as SOAPdenovo2 (Version 2.04: released on July 13th, 2012) software.

The method may further include removing low-quality data, invalid data and/or linker contamination data in the sequencing result after the whole genome of the transgenic organism is sequenced using a high-throughput sequencing platform to obtain clean data. The sequence splicing may be sequence splicing of the cleaning data.

In the above method, the sequence B may be a contig or a scaffold. The overlapping sequences are all known sequences obtained after sequence splicing, that is, there is no unknown sequence in the sequence. The framework sequence is a sequence in which some nucleotides (bases) are still unknown after sequence splicing.

In the above method, the transgenic organism can be a transgenic plant or a transgenic animal. The recipient organism can be a plant or an animal.

In the above, sequence alignment can be performed using sequence alignment software and/or modules. The sequence alignment software may be Clustal software, such as ClustalW.

In order to solve the above technical problems, the present invention also provides a method for determining the copy number of exogenous DNA fragments in a transgenic organism.

The method for determining the copy number of exogenous DNA fragments in the transgenic organism provided by the present invention includes: determining the exogenous DNA fragments in the transgenic organism by using the method for determining the sequence and/or insertion position and/or marginal sequence of the exogenous DNA fragment in the transgenic organism the sequence and/or insertion position and/or marginal sequence to determine the copy number of the exogenous DNA segment in the transgenic organism.

In the above method, the transgenic organism can be a transgenic plant or a transgenic animal.

In order to solve the above technical problems, the present invention also provides a system for determining the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in transgenic organisms.

The system for determining the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in transgenic organisms provided by the present invention includes at least two of the following b1)-b4):

b1) Reagents and/or instruments required for high-throughput sequencing;

b2) reagents and/or instruments required for amplification;

b3) sequence alignment software and/or modules;

b4) Sequence splicing software and/or modules.

The above system may also consist of only at least two of the above b1)-b4).

In the above system, the transgenic organism can be a transgenic plant or a transgenic animal.

For solving the above-mentioned technical problems, the present invention also provides any of the following applications:

P1) the application of the method for determining the sequence and/or the insertion position and/or the marginal sequence of the exogenous DNA fragment in the transgenic organism or the method for determining the copy number of the exogenous DNA fragment in the transgenic organism in biological breeding;

P2) the application of the method for determining the sequence and/or the insertion position and/or the marginal sequence of the exogenous DNA fragment in the transgenic organism or the method for determining the copy number of the exogenous DNA fragment in the transgenic organism in evaluating whether the transgenic organism is safe;

P3) application of the system in determining the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in transgenic organisms;

P4) application of the system in determining the copy number of exogenous DNA fragments in transgenic organisms;

P5) application of the system in biological breeding;

P6) Application of the system in evaluating the safety of genetically modified organisms.

In the above application, the transgenic organism may be a transgenic plant or a transgenic animal. The organism can be a plant or an animal.

The method of the present invention has the following advantages:

1) The species restriction can be broken through. When the sequence of the vector used in constructing the transgenic organism is known or only the sequence of the target gene used in constructing the transgenic organism is known, the actual insertion of the transgenic organism can be determined by a clear method of this method. Insertion sequence, insertion position and marginal sequence of the foreign DNA fragment;

2) Can not be restricted by the target gene used in constructing the transgenic organism, more extensively detect the transgenic ride-on effect of the non-target gene in the transgenic vector, and simultaneously determine the sequence inserted into the genome of the recipient organism in the vector;

3) The insertion position, marginal sequence and even copy number of foreign genes can be detected more quickly and accurately;

4) When performing high-throughput sequencing, due to the limited depth of sequencing coverage, unknown sequences may exist in the whole genome sequence of the transgenic organism, and the method of the present invention can detect the complete genome sequence of the transgenic organism. Insertion Sequence, Insertion Position, and Marginal Sequence.

Description of drawings

Figure 1 shows the alignment result of the target gene AtD-CGS expression cassette sequence in the transgenic vector and scaffold99334.

Figure 2 shows the results of aligning the AtD-CGS expression cassette sequence of the target gene in the transgenic vector with scaffold99334 (before filling in blank unknown bases) and scaffold99334.fill (after filling blank unknown bases) using ClustalW software.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The method for determining the sequence, insertion position and marginal sequence of exogenous DNA fragments in transgenic organisms of the present invention includes the following 1)-5):

1) using a high-throughput sequencing platform to sequence the whole genome of the transgenic organism to obtain the whole genome sequence of the transgenic organism;

2) Sequence A is compared with the whole genome sequence of the transgenic organism to determine the sequence, insertion position and marginal sequence of the exogenous DNA fragment in the transgenic organism;

Sequence A is a1), a2) or a3):

a1) the full-length sequence of the vector used in the preparation of the transgenic organism;

a2) the sequence of any DNA fragment containing the exogenous DNA fragment in the vector;

a3) The sequence of the exogenous DNA fragment or any fragment thereof.

3) Sequence A is compared with the complete genome sequence of the transgenic organism to obtain a sequence matching the sequence A in the complete genome sequence of the transgenic organism, and the sequence is named as sequence B;

4) Sequence B is compared with the whole genome sequence of the wild-type organism as the reference sequence, and the sequence, insertion position and marginal sequence of the exogenous DNA fragment in the transgenic organism are preliminarily determined;

5) Design primers according to the known sequence and reference sequence in sequence B, use the primers to amplify the genome of the transgenic organism, and further determine the sequence, insertion position and marginal sequence of the exogenous DNA fragment in the transgenic organism according to the sequence of the amplified product.

The method for determining the sequence, insertion position and marginal sequence of exogenous DNA fragments in the transgenic organism will be specifically described below by taking transgenic soybean as an example.

Example 1. Determination of sequence, insertion position and marginal sequence of exogenous DNA fragments in AtDCGS high methionine soybean

1. Preparation of AtDCGS high methionine soybean CGS-ZG11

Using the vector pGPTV-Bar-DCGS, the AtD-CGS (also known as AtDCGS) gene was transformed into the soybean variety Zigong Dong bean by Agrobacterium-mediated transgenic method, and the AtD-CGS high methionine soybean CGS-ZG11 (that is, the AtD-transformed soybean CGS-ZG11) was obtained. -CGS high methionine transgenic soybean line (Han Qingmei et al., Identification and genetic stability analysis of high methionine transgenic soybean, Chinese Journal of Oil Crops, 2015, 37(6):789-796)).

2. Determination of the sequence, insertion position and marginal sequence of exogenous DNA fragments in AtD-CGS high methionine soybean CGS-ZG11

1. Preparation of genomic DNA samples of AtD-CGS high methionine soybean CGS-ZG11

The genomic DNA of AtD-CGS high methionine soybean CGS-ZG11 was extracted using the plant genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

2. Genome resequencing of AtD-CGS high methionine soybean

Based on the HiSEQ2500 sequencing technology platform, the paired-end sequencing (Paired-End) method was used to construct a small fragment library for genome sequencing, and the original sequencing data was obtained. Then remove linker contamination and low-quality data, and obtain clean data. The standards for data removal are as follows: a. When the proportion of undetermined base information in single-end sequencing reads is greater than 10%, the pair of reads needs to be removed; b . When the number of bases with low quality, that is, the Phred value lower than 5, contained in the single-end sequencing read exceeds 50% of the length of the read, this pair of reads needs to be removed. The clean sequences and sequences containing the indeterminate base N in the genome resequencing data of ATCGS high methionine soybean are shown in Table 1.

Table 1. Genome resequencing data of AtDCGS high methionine soybean

library Clean Reads Proportion of high-quality Q30 sequences 500bp 159,451,342 89.57%

1) The genome was assembled using SOAPdenovo2 (Version 2.04: released on July 13th, 2012) software, and the relevant information is shown in Table 2.

Table 2. The assembly results of the genome of AtDCGS high methionine soybean

soybean reference genome size 978495272bp Genome assembly size (including N) 964714736bp Genome assembly size (without N) 885873140bp Number of Scaffolds 1249045 Average Scaffold Length 772bp Scaffold median length 127bp Scaffold longest length 422777bp Scaffold shortest length 100bp N50 Scaffold Length 20840bp Number of Contigs 1985644 Average length of Contig 469bp Contig median length 137bp Contig longest length 51715bp Contig shortest length 100bp N50 Contig length 2783bp

In Table 2, the soybean reference genome is the whole genome sequence of Williams 82, and the version number of the soybean whole genome sequence is Gmax_275_Wm82.a2.v1.

2) According to the sequence of the target gene AtD-CGS expression cassette in pGPTV-Bar-DCGS (seed-specific promoter (LegB4) - guide peptide (TP) - target gene (AtD-CGS) - terminator (TOCT)) , compare and analyze the Scaffold sequence, and find that the sequence of the AtD-CGS expression cassette of the target gene is specifically aligned to scaffold99334 (55746bp), as shown in Figure 1 (scaffo in Figure 1 means scaffold99334, DCGS- means the sequence of the AtD-CGS expression cassette of the target gene) shown, and the sequence in scaffold99334 that matches the AtDCGS expression cassette sequence of the target gene is located in the middle part of the scaffold99334 sequence.

3) Align the obtained scaffold99334 sequence to the soybean reference genome in Table 2, and find that scaffold99334 is located between chromosome 09 Chr09:39880882-39924565, and use ClustalW software to compare scaffold99334 with the reference sequence segment (chromosome 09 Chr09: 39880882-39924565) were carefully aligned, and it was found that the underlined exogenous fragment was inserted between Chr09:39917686-39917762, as shown in Figure 2.

4) Due to the depth of sequencing coverage, there is an unknown sequence in the insertion sequence of the exogenous fragment and the marginal sequence of the insertion position in scaffold99334 (Figure 2). Based on the sequences flanking the unknown sequence, primers CG-F and CG-R (as shown in Table 3) were designed to amplify the unknown sequence, and the unknown sequence was subjected to next-generation sequencing.

Table 3. Primers for amplifying unknown sequences

primer name sequence CG-F 5'-AGGGCTGCTAAAGGAAGCGGAACA-3' CG-R 5'-CGATGTAGTGGTTTGACGATGGTG-3' GT-F 5'-GCCTGAAAATGAGGAAGAAACA-3' GT-R 5'-CAATGAATCAACAACTCTCCTGGCG-3' GA-F 5'-ACACTCAACCCTATCTCGGGCTATT-3' GA-R 5'-GGTATCTTATGGCTGCTTGGAGTTG-3'

5) Align the sequence obtained by sequencing the unknown sequence with the soybean reference genome in Table 2, and clarify the insertion position and marginal sequence of the exogenous fragment in the ATCGS high methionine soybean. As shown in Figure 2, the exogenous fragment (italicized) was inserted into soybean chromosome 09 between Chr09:39917686-39917762, and the marginal sequences at both ends of the insertion site can be obtained according to the soybean reference sequence.

6 At the same time, clarify the details of exogenous fragments. As shown in Figure 2, the inserted exogenous fragment reaches 9343bp, as shown in sequence 1 in the sequence table, in sequence 1, the 171-423rd position is the NOS terminator sequence, the 494th-2305th position is the GUS gene sequence, and the 2350th position -3061 is the TOCT terminator sequence, 3132-4535 is the AtD-CGS gene sequence, 4719-7472 is the LegB4 promoter sequence, 7513-8125 is the pNOS promoter sequence, and 8126-8714 is the herbicide Resistance gene Bar gene sequence, the 8715-8937th is the transcript 7 terminator sequence. This exogenous fragment information is consistent with the target vector. At the same time, using the insertion position information, two pairs of primers (GT-F/GT-R and GA-F/GA-R (that is, GT-F/GT-R and GA-F/ GA-R)), carry out PCR amplification, and confirm the insertion position by conventional sequencing, indicating that the method for determining the sequence, insertion position and marginal sequence of exogenous DNA fragments in transgenic organisms of the present invention is used to determine the exogenous transfection in AtDCGS high methionine soybean. The results of DNA fragment sequence, insertion position and marginal sequence are reliable.

6) According to the sequence of the target gene AtD-CGS expression cassette in pGPTV-Bar-DCGS (seed-specific promoter (LegB4) - guide peptide (TP) - target gene (AtD-CGS) - terminator (TOCT)) , compared and analyzed the Scaffold sequences, and found that except scaffold99334, no other sequences that can be specifically aligned with the target gene expression cassette were detected, indicating that the target gene exists in a single copy in the genome of AtD-CGS high methionine soybean CGS-ZG11. This is consistent with the related Southern hybridization results (Han Qingmei et al., Identification and genetic stability analysis of high-methionine transgenic soybeans, Chinese Journal of Oil Crops, 2015, 37(6):789-796). It shows that this method also has the advantage of being able to detect the copy number.

Claims (7)

1. A method for determining the sequence and/or insertion position and/or marginal sequence of exogenous DNA fragments in a transgenic organism, including the following 1) and 2): 1) Sequence the whole genome of the transgenic organism to obtain the whole genome sequence of the transgenic organism; wherein, the transgenic organism is a transgenic organism obtained by processing the recipient organism with a vector containing exogenous DNA fragments; 2) Including the following 21) and 22): 21) Sequence alignment of sequence A with the whole genome sequence of the transgenic organism to obtain a sequence matching the sequence A in the whole genome sequence of the transgenic organism, and name the sequence as sequence B; 22) includes the following 22a) and 22b): 22a) aligning the sequence B with the reference sequence of the recipient organism, and preliminarily determining the sequence and/or the insertion position and/or the marginal sequence of the exogenous DNA fragment in the transgenic organism; 22b) Design primers according to the known sequence in the sequence B and the reference sequence of the recipient organism, use the primers to amplify the genome of the transgenic organism, and further determine the transgenic organism according to the sequence of the amplified product the sequence and/or insertion position and/or marginal sequence of the exogenous DNA fragment; Said sequence A is a1), a2) or a3): a1) the full-length sequence of the vector; a2) the sequence of any DNA fragment from the vector containing the exogenous DNA fragment; a3) The sequence of the exogenous DNA fragment or any fragment thereof. 2 . The method according to claim 1 , wherein the sequencing of the entire genome of the transgenic organism is performed using a high-throughput sequencing platform. 3 . 3. The method of claim 1, wherein the method further comprises constructing a library for sequencing the genome of the transgenic organism before sequencing the entire genome of the transgenic organism. 4. The method according to claim 2, characterized in that: after using a high-throughput sequencing platform to sequence the whole genome of the transgenic organism, sequence splicing is performed by sequence splicing software and/or modules to obtain the whole genome sequence of the transgenic organism. 5. The method according to claim 1, wherein the transgenic organism is a transgenic plant or a transgenic animal. 6. A method for determining the copy number of an exogenous DNA fragment in a transgenic organism, comprising: determining the sequence and/or insertion position and/or marginal sequence of the exogenous DNA fragment in the transgenic organism by utilizing the method described in any one of claims 1-5 The copy number of the exogenous DNA segment in the transgenic organism. 7. Any of the following applications: P1) Application of any one of the methods of claims 1-6 in biological breeding; P2) Application of the method of any one of claims 1 to 6 in evaluating whether a transgenic organism is safe.

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