CN114517239A - Detection method of transgenic soybean WYN029GmA - Google Patents

Abstract

The invention relates to the field of transgenic soybeans, in particular to a detection method of WYN029GmA of transgenic soybeans. The invention provides a transgenic soybean WYN029GmA transformation event exogenous insertion vector site, a left flank sequence and a right flank sequence thereof, and provides a method for detecting transgenic soybean. The scheme can quickly, efficiently and specifically detect whether the sample to be detected is derived from the transgenic soybean, and has important significance and wide application prospect in the aspects of detection of the transgenic soybean and identification of germplasm resources.

Description

Detection method of transgenic soybean WYN029GmA

Technical Field

The invention relates to the field of transgenic soybeans, in particular to a detection method of WYN029GmA of transgenic soybeans.

Background

Since the first case of transgenic crop approval commercialization in 1996, the global transgenic crop planting area has exceeded 1.9 hundred million hectares, increased 113-fold over 1996, and a total of 70 national applications. The transgenic soybean is the transgenic grain crop with the earliest industrialization, the highest application rate, the widest planting area and the largest growth speed, and occupies 50 percent of the area of the transgenic crop in the world. 8803 ten thousand tons of imported soybeans in 2018 are the largest consumption countries of transgenic soybeans, so the soybean production becomes more important and the development potential is huge. In soybean fields in China, the area of medium weeds reaches more than 50%, the soybean loss is about 15%, and glyphosate is used as a systemic biocidal herbicide, has the advantages of broad weeding spectrum, low toxicity, low residue and the like, and is the herbicide with the largest use amount at present. The application of the glyphosate-resistant transgenic soybean successfully solves the field weeding problem and is beneficial to the mechanized and large-scale production of the soybean.

The AM79-EPSPS gene is obtained by directly separating and extracting soil metagenome DNA from the soil polluted by glyphosate, constructing a genome library and utilizing an EPSPS function deletion mutant strain function complementation experiment, and is a new gene with independent intellectual property rights in China. The gene coding protein belongs to EPSPS, and is a brand new EPSPS protein. The AM79-EPSPS protein not only has higher glyphosate tolerance, but also keeps stronger affinity with phosphoenolpyruvate pyruvate (PEP), and can endow plants with the tolerance to herbicide glyphosate.

According to the development trend of global transgenic crops, more transgenic crops are promoted for commercial production in the future, the detection of the transgenic crops is required according to relevant national regulations, the existing method for detecting the transgenic soybeans mainly comprises polymerase chain reaction or probe hybridization of nucleotide sequences, exogenous promoters, terminators, marker genes and the like, different transformants cannot be distinguished, if the transgenic materials are subjected to whole genome sequences, high sequencing cost and long time period are required, and the preparation of antibodies and antigens required by the enzyme-linked immunosorbent assay at the protein level is difficult, so that a simple, convenient and quick detection method is required to identify specific transgenic events.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide a method for detecting transgenic soybean WYN029GmA, which can quickly, efficiently, and specifically detect whether a sample to be detected is derived from the above transgenic soybean, and has important significance and wide application prospects in detection of transgenic soybean and identification of germplasm resources.

The invention provides application of 20546065 locus on the No. 18 chromosome of soybean in transgenic soybean detection; the transgenic soybean comprises WYN029GmA transgenic soybean and/or soybean bred by using WYN029GmA as a parent.

The invention also provides a left flank nucleic acid of the exogenous gene insertion site of the transgenic soybean WYN029GmA, wherein the left flank nucleic acid has the nucleotide sequence shown as SEQ ID NO: 2 under the condition of high nucleotide sequence.

The invention provides right flank nucleic acid of a transgenic soybean WYN029GmA exogenous gene insertion site, wherein the right flank nucleic acid has a nucleotide sequence shown as SEQ ID NO: 3.

The invention also provides application of the left flank nucleic acid and/or the right flank nucleic acid as a marker in preparation of a transgenic soybean detection reagent; the transgenic soybean comprises WYN029GmA transgenic soybean and/or soybean bred by using WYN029GmA as a parent.

The invention provides a transgenic soybean detection kit, which comprises: primer pair A and/or primer pair B; in the primer pair A, an upstream primer A targets the left flanking nucleic acid; the downstream primer A targets T-DNA of mam79-pC 3301;

in the primer pair B, a downstream primer B targets the right flanking nucleic acid; the upstream primer B targets the T-DNA of mam79-pC 3301.

The invention provides the detection kit, and the primer pair A comprises two primers with nucleotide sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6.

The invention also provides a method for designing 3 pairs of PCR primer pairs of the left flank sequence according to the integration site information, then screening out a pair of primers with the characteristics of high specificity, good accuracy and the like, and designing 3 pairs of primer pairs with nucleotide sequences shown as SEQ ID NO:5 and SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10, and the screened primer pair is shown as SEQ ID NO:5 and SEQ ID NO: and 6.

The invention provides the detection kit, and the primer pair B comprises two primers with nucleotide sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12.

The invention also provides a method for designing 3 pairs of PCR primer pairs with right flank sequences according to the integration site information, then screening out a pair of primers with the characteristics of high specificity, good accuracy and the like, and designing 3 pairs of primer pairs with nucleotide sequences shown as SEQ ID NO:11 and SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: shown at 16. The screened primer pair is shown as SEQ ID NO:11 and SEQ ID NO: shown at 12.

One of the primer pairs is designed on a mam79 EPSPS gene of T-DNA, and the other primer pair is designed on a soybean genome on the right side of the T-DNA, so that during amplification, only a transgenic soybean with the T-DNA inserted into the 20546065 th nucleotide of the 18 th chromosome is taken as a template to obtain a target fragment, and other transgenic soybeans or non-transgenic soybeans are taken as templates to be incapable of obtaining the target fragment by amplification, so that whether a detection object is the transgenic soybean with the T-DNA inserted into the 20546065 th nucleotide of the 18 th chromosome can be easily judged.

The invention provides a PCR hybridization probe for detecting transgenic soybean, which is designed by the left flank sequence and/or the right flank sequence.

The invention provides a detection method of transgenic soybeans, which is used for detecting a soybean sample, wherein the transgenic soybeans comprise WYN029GmA transgenic soybeans and/or soybeans bred by using WYN029GmA as parents.

The soybean sample of the present invention comprises soybean seeds, tissues or organs of soybean plants and/or soybean products. The soy products of the present invention include food, seasoning and/or feed containing soy ingredients.

The detection method of the transgenic soybean provided by the invention comprises the steps of carrying out sequencing after PCR, wherein the sequencing result comprises a sequence shown by SEQ ID NO. 17 and/or SEQ ID NO. 18, and the sample contains the transgenic soybean and/or a soybean component bred by using WYN029GmA as a parent; the sequencing result does not comprise the sequence shown in SEQ ID NO. 12 and/or SEQ ID NO. 13, so that the sample does not contain transgenic soybean and/or soybean components bred by using WYN029GmA as a parent.

Wherein the sequence shown in SEQ ID NO. 17 is a part from the 5' end of the exogenous gene to the downstream primer of the primer pair A.

Wherein the sequence shown in SEQ ID NO. 18 is the part from the upstream primer of the primer pair B to the 3' end of the foreign gene.

Or the detection comprises electrophoresis after PCR, the band is 273bp and/or 300bp, and the sample contains transgenic soybeans and/or soybean components bred by using WYN029GmA as parents; if there is no band, the sample does not contain transgenic soybean and/or soybean components bred from WYN029GmA as parents.

The invention provides a transgenic soybean WYN029GmA transformation event exogenous insertion vector site, a left flank sequence and a right flank sequence thereof, and provides a method for detecting transgenic soybean. The scheme can quickly, efficiently and specifically detect whether the sample to be detected is derived from the transgenic soybean, and has important significance and wide application prospect in the aspects of detection of the transgenic soybean and identification of germplasm resources.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts according to the drawings:

FIG. 1 shows a map of the mam79-pC 3301T-DNA region;

FIG. 2 is a schematic illustration of an insertion position;

FIG. 3 shows the sequencing results of left border PCR, where the upper case part of the sequence is the flanking sequence of the insertion site and the lower case part is the transgene vector sequence;

FIG. 4 shows the sequencing results of right border PCR, where the upper case part of the sequence is the insertion site flanking sequence and the lower case part is the transgene vector sequence;

FIG. 5 shows the soybean genome Chr18: 20546065-20545967 sequences;

FIG. 6 shows a PCR detection electropherogram specific for the transformant WYN029GmA, wherein A: left border specific detection electropherograms; b: detecting the electrophoresis chart of the right border specificity; m: the molecular weight standard is 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp and 100bp from top to bottom; CK 1: water control; CK 2: plasmid control; CK 3: negative control wild type; 1-3: t2 generation transformant; 4-6: t3 generation transformant; 7-9: t4 generation transformant.

Detailed Description

The invention provides a detection method of transgenic soybean WYN029GmA, and a person skilled in the art can appropriately improve process parameters for reference by the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The connection region of each transgenic biological strain exogenous T-DNA and the plant genome has specificity, and the strain specificity detection method has very high specificity and accuracy, so that the specificity detection method established based on the flanking sequence of the exogenous T-DNA integration site in the transgenic event is an important technical means for realizing effective supervision and management of the transgenic event and derived varieties thereof and ensuring the healthy development of transgenic industry. The invention provides a detection method of transgenic soybean WYN029GmA, which can quickly, efficiently and specifically detect whether a sample to be detected is derived from the transgenic soybean.

The transgenic soybean WYN029GmA is obtained by partially inserting T-DNA of a plasmid mam79-pC3301 with a mam79 epsps gene into a soybean Tianlong I genome through an agrobacterium-mediated soybean cotyledonary node method, has better herbicide resistance and better application and breeding values, and obtains an environment release batch text at the end of 2019. However, before the invention, no one knows the specific insertion site of the T-DNA, and cannot establish a method for effectively identifying the transgenic soybean.

The invention extracts genome DNA to carry out TailPCR and sequencing to obtain a transgenic soybean WYN029GmA genome sequence. Analyzing the sequencing result, and screening 1 fragment related to the vector sequence as shown in SEQ ID NO: 1, shown in the specification. The splicing fragment was subjected to BLAST analysis at NCBI, and the insertion site of T-DNA was determined to be 20546065 th nucleotide on chromosome 18 based on the BLAST analysis result. The region into which the foreign gene is inserted is a non-coding region.

The exogenous insertion vector and the left flanking sequence of the transgenic soybean WYN029GmA transformation event cover partial T-DNA of a mam79-pC3301 plasmid and a soybean genome sequence on the left side of the T-DNA insertion position, and the transgenic soybean with the T-DNA sequence of mam79-pC3301 inserted at the 20546065 th nucleotide of the 18 th chromosome can be specifically characterized. Based on the sequence, whether a sample to be detected is derived from the transgenic soybean can be specifically detected, and the method has important significance and wide application prospect in the aspects of detection of the transgenic soybean and identification of germplasm resources.

In some embodiments, the use comprises designing a PCR primer pair or hybridization probe for detecting the transgenic soybean based on the left or right flank sequence.

One designed primer in the primer pair is arranged in a T-DNA sequence at one end, and the other end is arranged in a soybean genome: if the upstream primer is on the left border genome and the downstream primer is on the T-DNA; the upstream primer is on the T-DNA and the downstream primer is on the right border genome. Therefore, in the amplification, the target fragment can be amplified only by using the transgenic soybean in which the T-DNA is inserted into the 20546065 th nucleotide of the 18 th chromosome as a template, and the target fragment cannot be amplified by using other transgenic soybean or non-transgenic soybean as a template, so that whether the detection object is the transgenic soybean in which the T-DNA is inserted into the 20546065 th nucleotide of the 18 th chromosome can be easily judged.

The kit provided by the invention is characterized in that the DNA polymerase is selected from one or more of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase and Klenow fragment.

According to one kit provided by the invention, the DNA polymerase of the kit is TaqDNA polymerase which is cold start TaqDNA polymerase.

The invention also provides a using method of the kit, which comprises the steps of taking the DNA of a sample to be detected as a template, carrying out PCR amplification by taking the primer pair as an upstream primer and a downstream primer, and judging whether a T-DNA fragment of mam79-pC3301 is inserted into the 20546065 th nucleotide on the No. 18 chromosome of the sample according to a PCR amplification product.

The invention also provides a kit for PCR, which comprises the primer pair and an auxiliary reagent.

In some embodiments, the auxiliary reagents comprise one or more of water, DNA polymerase, dNTPs, PCR buffer, positive control, and negative control; preferably, the DNA polymerase is selected from one or more of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow fragment; more preferably, the DNA polymerase is taq DNA polymerase; most preferably, the Taq DNA polymerase is cold start Taq DNA polymerase.

The invention also provides a use method of the PCR primer pair or the kit, the method comprises the steps of carrying out PCR amplification by using the DNA of a sample to be detected as a template and the primer pair as an upstream primer and a downstream primer, and judging whether a T-DNA fragment of mam79-pC3301 is inserted at the 20546065 th nucleotide on the No. 18 chromosome of the sample or not according to a PCR amplification product.

The using method can detect whether the mam79 EPSPS gene is inserted into the 20546065 th nucleotide on the 18 th chromosome of the sample by a simple PCR method, and has the advantages of convenient detection, good specificity and good accuracy.

The invention provides the using conditions of the kit, wherein the using conditions comprise that the annealing temperature of PCR reaction is 52-58 ℃ and the cycle number is 30-35.

In some embodiments, the PCR reaction is performed at an annealing temperature of 56 ℃ to 60 ℃ and a cycle number of 35.

In other embodiments, the PCR reaction is performed at an annealing temperature of 56 ℃ and a cycle number of 35.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

Taking leaves of transgenic soybean WYN029GmA planted in a field, extracting genome DNA, and carrying out Tail PCR and sequencing to obtain a transgenic soybean WYN029GmA genome sequence. The transgenic soybean WYN029GmA is obtained by taking soybean Tianlong I as a receptor material and plasmid mam79-pC3301 as a transformation vector through an agrobacterium-mediated soybean cotyledonary node transformation method, and the map of the plasmid mam79-pC3301 is shown in figure 1 in the attached figure of the specification.

Example 2

The integration site of the exogenous T-DNA in the soybean genome was determined according to the following method: the sequencing results of example 1 were analyzed to screen 1 fragment (SEQ ID NO: 1) associated with the vector sequence. BLAST analysis was performed on the sequenced fragments at NCBI, and the T-DNA insertion site was initially determined on chromosome 18 of the soybean genome based on the BLAST analysis results.

Example 3 specific PCR assay for transgenic Soybean

According to the information of the integration site, flanking sequence verification primers are respectively designed at the upstream and the downstream of the integration site for verifying the separation result of the flanking sequence. Primers were in the T-DNA sequence at one end and in the soybean genome at the other end: if the left upstream primer is on the left border genome and the downstream primer is on the T-DNA; the right upstream primer is on the T-DNA and the downstream primer is on the right border genome. See table 1 for specific primer sequences. And verifying the sequencing result. And finally, sequencing the PCR product, analyzing the sequencing result by using NCBI (national center for Biotechnology information) to obtain a left boundary sequence (SEQ ID NO: 2) and a right boundary sequence (SEQ ID NO: 3), determining that the WYN029GmA transformant exogenous fragment is integrated on the soybean genome No. 18 chromosome, wherein the integration site is in Chr 18: 20546065-20545967 (FIG. 2).

As shown in fig. 5 soybean genome Chr 18: 20546065-20545967 (SEQ ID NO: 4), wherein the first half part shown with boxes is matched with the sequence of the lower case letter part of the left border sequencing result (figure 3), and the second half part shown with shaded parts is matched with the sequence of the lower case letter part of the right border sequencing result (figure 4), and the results show that the integrated regions of the T-DNA positioned by the left and right flanking sequences in the soybean genome are completely consistent, and the transgenic event is a single-copy transgenic event. During the process of integrating the T-DNA into the soybean genome, the left and right border sequences of the vector are deleted, 193bp (including 26bp left border sequence, 84bp spacer sequence and 83bp promoter sequence) is deleted in the left region of the T-DNA insertion sequence, and 48bp (including 22bp spacer sequence and 26bp right border sequence) is deleted in the right region.

Table 1: transformation event-specific PCR detection of exogenous insert-related parameters

2. The primers are used for PCR detection and screening out a primer pair with high specificity and accuracy, wherein the reaction conditions are as follows:

step 1: 5min at 95 DEG C

Step 2: 30s at 95 DEG C

And 3, step 3: at 56 ℃ for 30s

And 4, step 4: 30s at 72 DEG C

And 5, step 5: 72 ℃ for 10min

And the 2 nd to 4 th steps are circulated for 35 times.

3. The amplified product was detected by 2% agarose gel, and after screening, the screened primer pair could not amplify to band in both plasmid and negative material, and only the positive material of transgene obtained the segment with expected size (FIG. 6).

4. Selecting 3 samples of transgenic plant and non-transgenic plant leaves from a test field, respectively extracting genome DNA, carrying out PCR detection by adopting screened primers, and displaying according to an electrophoresis result that target bands can be detected from the 3 transgenic leaf samples, the sizes of the target bands are 273bp and/or 300bp, no band is detected from the 3 non-transgenic leaf samples, and the result is negative.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaggctattt ctccgccacc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cattcgcatc gtggaccctg 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tggggactac acaggagaga 20

<210> 17

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ctcaatgaca agaagaaaat cttcgtcaac atggtggagc acgacacact tgtct 55

<210> 18

<211> 224

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 60

taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 120

atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 180

cgcggtgtca tctatgttac tagatcggga attaaactat cagt 224

Claims (10)

1. Application of 20546065 th site on 18 th chromosome of soybean in transgenic soybean detection; the transgenic soybean comprises WYN029GmA transgenic soybean and/or soybean bred by using WYN029GmA as a parent.

2. A left flank nucleic acid of a foreign gene insertion site of transgenic soybean WYN029GmA, wherein the left flank nucleic acid has the nucleotide sequence as set forth in SEQ ID NO: 2.

3. Right flank nucleic acid of a transgenic soybean WYN029GmA exogenous gene insertion site, wherein said right flank nucleic acid has the sequence as set forth in SEQ ID NO: 3.

4. Use of the left flanking nucleic acid of claim 2 and/or the right flanking nucleic acid of claim 3 as a marker for the preparation of a transgenic soybean detection reagent; the transgenic soybean comprises WYN029GmA transgenic soybean and/or soybean bred by using WYN029GmA as a parent.

5. The kit for detecting the transgenic soybean is characterized by comprising: primer pair A and/or primer pair B;

in the primer pair A, an upstream primer A targets the left flanking nucleic acid of claim 2; the downstream primer A targets T-DNA of mam79-pC 3301;

in the primer pair B, a downstream primer B targets the right flank nucleic acid of claim 3; the upstream primer B targets the T-DNA of mam79-pC 3301.

6. The detection kit according to claim 5, wherein the primer pair A comprises two primers having nucleotide sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6.

7. The detection kit according to claim 5, wherein the primer pair B comprises two primers having nucleotide sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12.

8. The method for detecting transgenic soybean, characterized in that the transgenic soybean detection kit of claim 5 is used for detecting soybean samples, wherein the transgenic soybean comprises WYN029GmA transgenic soybean and/or soybean bred by using WYN029GmA as a parent.

9. The soybean sample of claim 8, wherein the soybean sample comprises a tissue or organ and/or a soybean product taken from a soybean seed, a soybean plant.

10. The detection method according to claim 8,

the detection comprises sequencing after PCR, and the sequencing result comprises a sequence shown by SEQ ID NO. 17 and/or SEQ ID NO. 18, so that the sample contains transgenic soybean and/or soybean components bred by using WYN029GmA as a parent; the sequencing result does not comprise the sequence shown in SEQ ID NO. 17 and/or SEQ ID NO. 18, so that the sample does not contain transgenic soybean and/or soybean components bred by using WYN029GmA as a parent;

or the detection comprises electrophoresis after PCR, the band is 273bp and/or 300bp, and the sample contains transgenic soybeans and/or soybean components bred by using WYN029GmA as parents; if there is no band, the sample does not contain transgenic soybean and/or soybean components bred from WYN029GmA as parents.

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