CN114517239B - 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 transgenic soybeans WYN029 GmA. The invention provides a transgenic soybean WYN029GmA transformation event exogenous insertion vector site and left and right flanking sequences thereof, and provides methods for detecting transgenic soybean. The scheme can rapidly, 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 transgenic soybeans WYN029 GmA.

Background

The first transgenic crop in 1996 is approved for commercialization, the global transgenic crop planting area is over 1.9 hundred million hectares, and the planting area is increased by 113 times compared with 1996, and the application of the transgenic crop is 70 or more countries. Transgenic soybean is the earliest in industrialization, highest in application rate, widest in planting area and largest in growth speed, and accounts for 50% of the area of transgenic crops worldwide. In 2018, china imports soybean 8803 ten thousand tons, which is the largest consumer country of transgenic soybean, so the soybean production becomes particularly important and has huge development potential. In soybean fields in China, the area of moderate grass damage reaches more than 50%, soybean loss is about 15%, and glyphosate serving as a systemic herbicide has the advantages of broad-spectrum weeding, 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 soil polluted by glyphosate, constructing a genome library and utilizing the functional complementation experiment of EPSPS functional deletion mutant strain, and is a novel gene with independent intellectual property rights of 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 phosphoenolpyruvic acid (PEP), and can endow plants with tolerance to herbicide glyphosate, and the mam79-EPSPS gene used in the invention is obtained by artificially optimizing and modifying the AM79-EPSPS gene according to the codon preference of soybeans, so that the method is an excellent choice for cultivating glyphosate-resistant transgenic soybeans.

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

Disclosure of Invention

In view of the above, the technical problem to be solved by the invention is to provide a method for detecting transgenic soybean WYN029GmA, which can rapidly, efficiently and specifically detect whether a 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.

The invention provides an application of 20546065 locus on soybean chromosome 18 in transgenic soybean detection; the transgenic soybean comprises WYN029GmA transgenic soybean and/or soybean bred by WYN029GmA serving as a parent.

The invention also provides a left flanking nucleic acid of an exogenous gene insertion site of transgenic soybean WYN029GmA, which has a nucleotide sequence as set forth in SEQ ID NO:2, and a nucleotide sequence shown in the following formula.

The invention provides a right flanking nucleic acid of an exogenous gene insertion site of transgenic soybean WYN029GmA, which has a nucleotide sequence as shown in SEQ ID NO:3, and a nucleotide sequence shown in 3.

The invention also provides application of the left flank nucleic acid and/or the right flank nucleic acid serving as a marker in preparing a transgenic soybean detection reagent; the transgenic soybean comprises WYN029GmA transgenic soybean and/or soybean bred by WYN029GmA serving 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; downstream primer A targets the T-DNA of mam79-pC 3301;

in the primer pair B, a downstream primer B targets the right flank 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 3 pairs of PCR primer pairs of the left flanking sequence according to the integration site information, and then a pair of primers with the characteristics of high specificity, good accuracy and the like are screened out, wherein the nucleotide sequences of the 3 pairs of designed primers are 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, the screened primer pair is shown as SEQ ID NO:5 and SEQ ID NO: shown at 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 of the right flank sequence according to the integration site information, and then screening a pair of primers with the characteristics of high specificity, good accuracy and the like, wherein the nucleotide sequences of the 3 pairs of designed primer pairs are 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 the mam79 EPSPS gene of the T-DNA and the other is designed on the soybean genome on the right side of the T-DNA, so that during amplification, only the transgenic soybean inserted into the 20546065 nucleotide of the 18 # chromosome is used as a template to amplify the target fragment, and other transgenic soybean or non-transgenic soybean is used as a template to not amplify the target fragment, thereby easily judging whether the detected object is the transgenic soybean inserted into the 20546065 nucleotide of the 18 # chromosome.

The invention provides PCR hybridization probes designed for detecting transgenic soybeans by the left flanking sequence and/or the right flanking sequence.

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

The soybean samples of the present invention include soybean seeds, tissues or organs of soybean plants, and/or soybean products. The soybean product of the present invention includes foods, condiments and/or feeds containing soybean components.

The detection method of the transgenic soybean provided by the invention comprises the steps of sequencing after PCR, wherein the sequencing result comprises sequences shown in SEQ ID NO. 17 and/or SEQ ID NO. 18, and the sample contains the 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. 12 and/or SEQ ID NO. 13, which shows 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 primer downstream 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 exogenous gene.

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

The invention provides a transgenic soybean WYN029GmA transformation event exogenous insertion vector site and left and right flanking sequences thereof, and provides methods for detecting transgenic soybean. The scheme can rapidly, 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

For a clearer description of embodiments of the invention or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the invention, and that, without the inventive effort, other drawings can be obtained from them to those skilled in the art:

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

FIG. 2 shows a schematic view of an insertion position;

FIG. 3 shows the sequencing results of left border PCR wherein the uppercase portion of the sequence is flanking the insertion site and the lowercase portion is the transgene vector sequence;

FIG. 4 shows the sequencing results of right border PCR wherein the uppercase portion of the sequence is flanking the insertion site and the lowercase portion is the transgene vector sequence;

FIG. 5 shows soybean genome Chr18:20546065 ～ 20545967 sequence;

FIG. 6 shows an electrophoresis diagram of transformant WYN029 GmA-specific PCR detection wherein A: detecting an electrophoresis chart of left boundary specificity; b: right boundary specificity detection electropherograms; m: molecular weight standard, which is 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom; CK1: water control; CK2: plasmid control; CK3: negative control wild type; 1 to 3: t2 generation transformants; 4 to 6: t3 generation transformants; 7-9: t4 generation transformants.

Detailed Description

The invention provides a detection method of transgenic soybean WYN029GmA, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

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

Transgenic soybean WYN029GmA was obtained by inserting the T-DNA portion of plasmid mam79-pC3301 carrying the mam79 epsps gene onto the genome of Soy Tianlong No. 1 by the Agrobacterium-mediated soybean cotyledonary node method, which has good herbicide resistance, good application and breeding value, and has obtained environmental release wholesale in the 2019 year old. However, prior to the present invention, the specific insertion site of the T-DNA was not known, and a method for efficiently identifying the transgenic soybean was not established.

The invention extracts genome DNA to carry out TailPCR and sequencing, and obtains the genome sequence of transgenic soybean WYN029 GmA. Analyzing the sequencing result, screening out 1 fragment related to the vector sequence as shown in SEQ ID NO:1, shown. BLAST analysis of the spliced fragment was performed on NCBI, and it was determined that the insertion site of T-DNA was at nucleotide 20546065 on chromosome 18 based on the result of BLAST analysis. The region into which the foreign gene is inserted is a non-coding region.

The exogenous insertion vector of the transgenic soybean WYN029GmA transformation event and the left flanking sequence cover the T-DNA of the mam79-pC3301 plasmid part and the soybean genome sequence at the left side of the T-DNA insertion part, and can specifically characterize the transgenic soybean with the T-DNA sequence inserted into mam79-pC3301 at the 20546065 th nucleotide of chromosome 18. Based on the sequence, whether the 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 flanking sequence or right flanking sequence.

One designed primer of the primer pair is provided with one end in a T-DNA sequence and the other end in a soybean genome: upstream primer on left border genome and downstream primer on T-DNA; the upstream primer is on the T-DNA and the downstream primer is on the right border genome. Therefore, in the case of amplification, the target fragment can be obtained by amplifying only the transgenic soybean having the T-DNA inserted into nucleotide 20546065 of chromosome 18 as a template, and the target fragment cannot be obtained by amplifying other transgenic soybean or non-transgenic soybean as a template, so that it is possible to easily determine whether or not the test object is the transgenic soybean having the T-DNA inserted into nucleotide 20546065 of chromosome 18.

The kit provided by the invention, wherein the DNA polymerase is selected from one or more of Taq, bst, vent, phi, pfu, tru, tth, tl, tac, tne, tma, tih, tf1, pwo, kod, sac, sso, poc, pab, mth, pho, ES4DNA polymerase and Klenow fragment.

The DNA polymerase of the kit is Taq DNA polymerase, and the kit is cold start Taq DNA polymerase.

The invention also provides a using method of the kit, which comprises the steps of taking DNA of a sample to be detected as a template, carrying out PCR amplification by using the primer pair as an upstream primer and a downstream primer, and judging whether a T-DNA fragment of mam79-pC3301 is inserted into a 20546065 nucleotide position on a 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 auxiliary reagents.

In some embodiments, the auxiliary reagent comprises 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, phi, 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 a cold start Taq DNA polymerase.

The invention also provides a using method of the PCR primer pair or the kit, which comprises the steps of carrying out PCR amplification by taking 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 into a 20546065 nucleotide position on a chromosome 18 of the sample according to a PCR amplification product.

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

The invention provides the use conditions of the kit, wherein the use conditions comprise the annealing temperature of the PCR reaction between 52 and 58 ℃ and the cycle number between 30 and 35.

In some embodiments, the annealing temperature of the PCR reaction is 56-60℃and the cycle number is 35.

In other embodiments, the annealing temperature of the PCR reaction is 56℃and the cycle number is 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 for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

The leaves of transgenic soybean WYN029GmA planted in the field are taken, genomic DNA is extracted for Tail PCR and sequencing, and the genomic sequence of the transgenic soybean WYN029GmA is obtained. The transgenic soybean WYN029GmA is obtained by using soybean Tianlong No. I as a receptor material, using a plasmid mam79-pC3301 as a transformation vector and adopting an agrobacterium-mediated soybean cotyledonary node transformation method, wherein the map of the plasmid mam79-pC3301 is shown in figure 1 of the specification.

Example 2

The integration site of the exogenous T-DNA in the soybean genome was determined as follows: the sequencing results in 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 soybean genome chromosome 18 based on the BLAST analysis results.

EXAMPLE 3 transgenic Soybean specific PCR detection

Flanking sequence verification primers are designed upstream and downstream of the integration site respectively for flanking sequence isolation result verification according to the integration site information. Primers are at one end in the T-DNA sequence and at the other end in the soybean genome: 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. The specific primer sequences are shown in Table 1. And verifying the sequencing result. Finally, the PCR product is sent to be sequenced, and the sequencing result is analyzed by NCBI to obtain a left border sequence (SEQ ID NO: 2) and a right border sequence (SEQ ID NO: 3), and the exogenous fragment of the WYN029GmA transformant is integrated on the soybean genome No. 18 chromosome, wherein the integration site is formed in the Chr18:20546065 ～ 20545967 (fig. 2).

Soybean genome Chr18 as in fig. 5: 20546065 ～ 20545967 sequence (SEQ ID NO: 4), the first half of the boxed representation matches the lower case sequence of the left border sequencing result (FIG. 3), the second half of the shaded representation matches the lower case sequence of the right border sequencing result (FIG. 4), these results indicate that the integration regions of the left and right flanking side sequence-located T-DNA in the soybean genome are completely identical, and the transgenic event is a single copy transgenic event. During integration of the T-DNA into the soybean genome, both the left and right border sequences of the vector are deleted, the left region of the T-DNA insert sequence is deleted by 193bp (including 26bp left border sequence, 84bp spacer sequence, 83bp promoter sequence) and the right region is deleted by 48bp (including 22bp spacer sequence, 26bp right border sequence).

Table 1: transformation event specific PCR detection-related parameters for exogenous inserts

2. PCR detection is carried out by using the primers, and a primer pair with high specificity and accuracy is screened, wherein the reaction conditions are as follows:

step 1: 95 ℃ for 5min

Step 2: 95 ℃ for 30s

Step 3: 30s at 56 DEG C

Step 4: 72 ℃ for 30s

Step 5: 72 ℃ for 10min

Step 2-4, the cycle is 35 times.

3. The amplified products were detected using a 2% agarose gel and screened, the screened primer pairs failed to amplify to bands on both plasmid and negative material, and only the transgene positive material gave fragments of the expected size (FIG. 6).

4. 3 samples of transgenic plants and non-transgenic plant leaves are selected from a test field, genomic DNA is extracted respectively, PCR detection is carried out by using the screened primers, according to electrophoresis results, the 3 transgenic leaf samples can detect target bands, the sizes of the target bands are 273bp and/or 300bp, and the 3 non-transgenic leaf samples do not detect the bands, so that the result is negative.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Sequence listing

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<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 (6)

1. A kit for detecting transgenic soybean, comprising: primer pair a and primer pair B;

in the primer pair A, an upstream primer A targets a primer sequence shown in SEQ ID NO:2, a left flanking nucleic acid; downstream primer A targets the T-DNA of mam79-pC 3301;

in the primer pair B, the downstream primer B targets the primer shown in SEQ ID NO:3, right flanking nucleic acid; the upstream primer B targets the T-DNA of mam79-pC 3301.

2. The detection kit according to claim 1, wherein the primer pair A comprises two primers having nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6.

3. The detection kit according to claim 1, wherein the primer pair B comprises two primers having nucleotide sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12.

4. The method for detecting transgenic soybeans is characterized in that a soybean sample is detected by using the transgenic soybean detection kit according to any one of claims 1-3, wherein the transgenic soybeans comprise WYN029GmA transgenic soybeans and/or soybeans bred by taking WYN029GmA as a parent.

5. The method of claim 4, wherein the soybean sample comprises tissue or organ from soybean seeds, soybean plants and/or soybean products.

6. The method according to claim 4, wherein,

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

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