CN107557359B - Right flank sequence of exogenous insertion vector for transgenic soybean WH8055 transformation event - Google Patents

Abstract

The invention relates to a right flank sequence of a transgenic soybean WH8055 transformation event exogenous insertion vector, application of the right flank sequence, a primer pair, a PCR kit and a use method of a primer and the PCR kit, wherein the nucleotide sequence of the right flank sequence is shown in SEQ ID NO. 4, and can specifically indicate transgenic soybeans with a T-DNA fragment of pCHA L1 inserted at the 43998179 th nucleotide of a No. 3 chromosome.

Description

Right flank sequence of exogenous insertion vector for transgenic soybean WH8055 transformation event

Technical Field

The invention relates to the field of transgenic soybeans, and in particular relates to a right flank sequence of a transgenic soybean WH8055 transformation event exogenous insertion vector.

Background

Soybean is a major oil crop in the world. The planting area of the global transgenic crops in 2015 is up to 1.797 hundred million hectares, wherein the planting area of the soybeans is 0.916 million hectares and accounts for 51 percent of the total area^[1]. China is not only a large country for agricultural production, but also a large country for agricultural consumption. Since 1996, china has become a net importer of soybean. With the increasing domestic demand, the import quantity of transgenic soybean is also inIncreasing year by year. In 2015 years only, the import of Chinese transgenic soybean is up to 8169 ten thousand tons, and the year-by-year growth is 14.4%^[2]。

Scientific research and evaluation on the biosafety problem of the transgenic soybean is an important technical basis for supervising and managing the transgenic soybean and ensuring the healthy development of the transgenic soybean, and the transgenic detection technology is one of the key technologies for biosafety evaluation^[3]Has great significance for the identity verification of the transgenic plant, and is helpful for researching the expression mechanism of the exogenous gene and the influence of the exogenous gene in the receptor genome, thereby being helpful for evaluating the biological safety of the transgenic plant^[4]。

At present, some HA L transgenic plants exist, but HA L1 transgenic soybeans are not obtained through research, and a method capable of effectively detecting HA L1 transgenic soybeans is further lacked.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first objective of the present invention is to provide a right flank sequence of a transgenic soybean WH8055 transformation event foreign insertion vector, which can specifically indicate a transgenic soybean with a T-DNA fragment of pCHA L1 inserted at nucleotide 43998179 on chromosome 3.

The second purpose of the invention is to provide an application of the right flanking sequence in detecting transgenic soybean, and the application can accurately judge whether the sample is from the transgenic soybean with a T-DNA fragment of pCHA L1 inserted at nucleotide 43998179 on chromosome 3 through the right flanking sequence.

The third purpose of the invention is to provide a PCR primer pair designed according to the right flanking sequence, the primer pair can rapidly and accurately detect whether a T-DNA fragment of pCHA L1 is inserted at the 43998179 th nucleotide on the No. 3 chromosome of the sample through PCR reaction, and the PCR primer pair has the advantages of convenient detection, good specificity and high accuracy.

The fourth purpose of the invention is to provide a PCR detection kit, which integrates the primer pair with other auxiliary reagents and has the advantage of convenient use.

The fifth object of the present invention is to provide a method for using the primer set or the kit, by which whether a T-DNA fragment of pCHA L1 is inserted at nucleotide 43998179 on chromosome 3 of a sample can be rapidly and accurately detected.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the right flank sequence of the exogenous insertion vector of the transgenic soybean WH8055 transformation event has a nucleotide sequence shown in SEQ ID NO:4, respectively.

The invention is based on transgenic soybean WH8055 provided by Jilin province agricultural science institute biology, wherein the transgenic soybean WH8055 is obtained by inserting T-DNA of plasmid pCHA L1 with HA L1 gene into genome of soybean Williams 82 through an agrobacterium-mediated soybean cotyledonary node method, and HAs good salt tolerance, good agronomic character, good application prospect and breeding value.

The invention elaborately designs 3 ' SP1, 3 ' SP2 and 3 ' SP3 sequences, combines a Genome Walkingkit kit of Takara company, and obtains a right flank sequence of a transgenic soybean WH8055 transformation time exogenous insertion vector through TAI L-PCR and sequencing alignment.

The nucleotide sequence of the right flanking sequence is shown as SEQ ID NO. 4, and comprises an HA L1 gene sequence of T-DNA on pCHA L1, a NOs 3' UTR sequence and a soybean genome sequence on the right side of the T-DNA, so that the right flanking sequence comprises a T-DNA sequence inserted into a vector and a soybean genome sequence on the right side of an insertion site, only a transgenic soybean inserted with pCHA L1T-DNA at the 43998179 position of a No. 3 chromosome HAs the right flanking sequence, and pCHA L1 and non-transgenic soybeans or other transgenic soybeans do not have the right flanking sequence.

Therefore, the right flank sequence can specifically indicate transgenic soybeans with pCHA L1T-DNA inserted at 43998179 th site of chromosome 3, particularly transgenic soybeans WH8055 or transgenic soybeans transformed by the transgenic soybeans WH 8055.

The invention also relates to application of the right flanking sequence in detecting transgenic soybean, wherein a T-DNA fragment of pCHA L1 is inserted into the 43998179 th nucleotide of chromosome 3 of the transgenic soybean, and preferably the transgenic soybean is WH8055 or the transgenic soybean transformed by the WH 8055.

The application of the invention is based on the right flank sequence, and the target transgenic soybean can be subjected to specificity detection by molecular biological methods, such as PCR and molecular hybridization, and the invention has the advantages of simple operation, high accuracy and the like.

In some embodiments, the PCR primer pair or hybridization probe for detecting the transgenic soybean is designed based on the right flanking sequence described above.

The invention also relates to a PCR primer pair designed by the right flank sequence, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO: 6 and SEQ ID NO: shown at 7.

The primer pair is respectively designed on the T-DNA and the soybean genome on the right side of the T-DNA insertion site, and can be used for amplifying to obtain a product of about 700bp only by taking a target transgenic soybean as a template, while the PCR primer pair cannot be used for amplifying to obtain the target product by taking a pCHA L1, a non-transgenic soybean or other varieties of transgenic soybeans as templates.

The invention also relates to a PCR detection kit, which comprises the primer pair and other reagents.

The kit integrates the primer pair with other auxiliary reagents used for PCR, and has the advantage of convenient use.

In some embodiments, the additional reagents include one or more of DNA polymerase, dNTPs, PCR reaction buffer, positive control, and negative control.

In some embodiments, 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.

Preferably, the DNA polymerase is Taq DNA polymerase.

More preferably, the Taq DNA polymerase is hot start Taq DNA polymerase.

In some embodiments, the additional reagents comprise a pre-mix of the PCR reaction, the pre-mix being a mixture of DNA polymerase, dNTPs, and PCR reaction buffer.

The invention also relates to a use method of the primer pair or the kit, which comprises the following steps:

(1) taking the DNA of a soybean sample to be detected as a template, and carrying out PCR amplification by using the primer pair or the primer pair in the kit and other reagents;

(2) and judging whether a T-DNA fragment of pCHA L1 is inserted at the 43998179 th nucleotide on the No. 3 chromosome of the soybean to be detected or not according to the PCR amplification product.

The application method can detect whether the HA L1 gene is inserted into the 43998179 th nucleotide on the No. 3 chromosome of the sample by a simple PCR method, and HAs the advantages of convenient detection, good specificity and good accuracy.

In some specific embodiments, the annealing temperature of the PCR reaction is 49-51 ℃, and the number of cycles is 28-32; preferably, the annealing temperature is 50 ℃ and the number of cycles is 30.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention relates to a transgenic soybean WH8055, wherein a T-DNA sequence on pCHA L is inserted into a 43998179 th nucleotide position on a No. 3 chromosome of the soybean, so that an HA L gene is obtained, and the transgenic soybean can resist salt and alkali, shows good agronomic characters and HAs wide application prospect.

(2) The primer pair is finally determined by elaborate design and repeated screening, is respectively designed on the soybean genomes flanking T-DNA and T-DNA, only the transgenic soybean with the T-DNA inserted into 43998179 th nucleotide of No. 3 chromosome is taken as a template to amplify to obtain a target fragment, and other templates cannot amplify to obtain the target fragment, so that whether a detection object is the T target transgenic soybean can be easily judged. In addition, the primer pair disclosed by the invention also has the advantages of good specificity and accuracy and high detection efficiency.

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.

FIG. 1 is a T-DNA map of the pCHA L1 plasmid, wherein the T-DNA includes a left border sequence (L B), a Bar gene, HA L1 gene, and a Right Border (RB);

FIG. 2 shows the results of the Tail-PCR electrophoresis of the right border, wherein M1: λ -Hind III digest, 1-3: AP 11 st 2nd3rd, 4-6: AP 21 st 2nd3rd, 7-9: AP 31 st 2nd3rd, 10-12: AP 41 st 2nd3rd, 13-15: positive control 1st 2nd3rd, M2: D L2000;

FIG. 3 shows the sequencing result of TAI L-PCR at the right border, in which the shaded portion is the known sequence of HA L1 gene, the boxed portion is nos 3' UTR sequence, and the italic portion is soybean genome sequence;

FIG. 4 is the soybean genomic Chr03:43998179..43998678 sequence;

FIG. 5 is a diagram of the right border primer position of transgenic soybean WH8055, in which 3 'F is designed on T-DNA and 3' R is designed on the soybean genome to the right of the insertion site;

fig. 6 is an electrophoresis result diagram of specific PCR detection of transgenic soybean WH8055, wherein, M: marker, 1 is PCR reaction product of transgenic soybean WH 8055.

Detailed Description

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 manufacturers, and are all conventional products available on the market.

Example 1 acquisition of right flank sequence of transgenic Soybean WH8055 insertion vector

First, experimental material

A transgenic soybean WH8055 (provided by a soybean subject group of the institute of agriculture and sciences, Jilin province) is obtained by an agrobacterium-mediated soybean cotyledonary node transformation method (namely, a transgenic soybean WH8055 transformation event) by taking soybean Williams 82 as a receptor material and plasmid pCHA L1 as a transformation vector, wherein the map of the plasmid pCHA L1 is shown in a figure 1 in the specification, the transgenic soybean WH8055 shows saline-alkali resistance and good agronomic traits, and the insertion site of T-DNA is finally determined to be the 43998179 th nucleotide of the 3rd chromosome of soybean through example 1.

Second, acquisition of Right flank sequence

1. Extraction of DNA

(1) Collecting samples: leaves of transgenic soybean WH8055 planted in the field were harvested in the middle 6 th month and stored at-80 ℃.

(2) Extraction of DNA: the genomic DNA of the soybean leaf is extracted by CTAB method, and the specific detection method is shown in reference 5.

(3) Detection of DNA concentration and purity: and (3) detecting the concentration and purity of the DNA sample obtained in the step (2) by using Nano Drop.

2. Acquisition of the right flanking sequence

In this example, a Genome Walking Kit of TaKaRa company is used to obtain a flanking sequence of the right border of the exogenous T-DNA integration site in transgenic soybean WH 8055.

(1) 3 right border specific primers 3 ' SP1(SEQ ID NO: 1), 3 ' SP2(SEQ ID NO: 2) and 3 ' SP3(SEQ ID NO: 3) were designed based on the known HA L1 sequence (Table 1).

TABLE 1 Right Border specific primers

(2) Using 1. mu.l (500ng) of transgenic soybean genomic DNA as a template, AP1, AP2, AP3 and AP4 in 3 ' SP1, 3 ' SP2, 3 ' SP3 and TaKaRa Genome Walking Kit (Code No.6108) kits, and ck primers, 5 times were performed according to the TaKaRagenome Walking Kit (Code No.6108) Kit instructions, each 3 TAI L-PCR reactions.

(3) FIG. 2 shows that the 3rd PCR reactions ( lanes 3, 6 and 9) with primers AP1, AP2 and AP3 all gave specific bands that were clearly opposite to the control provided by the kit, and the bands (lane 12, size about 2.4kp) of the amplification products of 3 'AP 4-3rd of the same primers were cut and recovered and sequenced, i.e., the right flank sequence inserted into the vector pCHA L1 was obtained, the specific nucleotide sequence (SEQ ID NO:4) of the right flank sequence is shown in FIG. 3, wherein the shaded part is the known HA L1 gene sequence, the framed part is the NOs 3' UTR sequence, and the italic part is the soybean genome sequence.

3. Determination of integration site of exogenous T-DNA in soybean genome

The right sequence at the integration site of the exogenous T-DNA in transgenic soybean WH8055 was aligned with the soybean genome at the soybase.org website (https:// www.soybase.org/gb2/gbrowse/gmax2.0/) and was found to be present contiguously on the soybean genome Chr03:43998179..43998678(SEQ ID NO: 5) (FIG. 4), i.e.the integration site of the T-DNA in the soybean genome was obtained: the soybean genome Chr03:43998179 (i.e., at nucleotide 43998179 on chromosome 3 of soybean).

Example 2 specific PCR detection of transgenic Soybean WH8055

1. Design of PCR primers

Primer pairs capable of specifically detecting T-DNA insertion events were designed based on the right border flanking sequence of transgenic soybean WH8055 obtained in example 1 and the T-DNA sequence.

One primer of the primer pair is on the T-DNA, and the other primer is on the soybean genome: for example, the 3 'F forward primer was on the T-DNA and the 3' R reverse primer was on the right border genome (FIG. 5). The sequences of the primer pair 3 'F (SEQ ID NO: 6) and 3R' (SEQ ID NO: 7) are shown in Table 2.

TABLE 2 specific PCR detection primer pairs for transgenic soybean WH8055

2. Specific PCR detection of transgenic soybean WH8055

And (3) carrying out PCR detection by using the PCR primer pair by taking the genome DNA of the transgenic soybean WH8055 as a template. Wherein, the specific reaction conditions are as follows:

step 1: 94 ℃ for 2 min;

step 2: 30s at 94 ℃;

and 3, step 3: 30s at 50 ℃;

and 4, step 4: 30s at 72 ℃;

and 5, step 5: 5min at 72 ℃;

and 6, step 6: 1h at 16 ℃;

the 2nd to 4 th steps are circulated for 30 times.

3. The amplified products were detected by 1% agarose gel, and neither the plasmid nor the negative material could be amplified to bands, and only the transgene positive material obtained fragments of the expected size (FIG. 6).

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.

Reference documents:

[1] global Biotechnology/transgenic crop commercial development situation of global source james.2015 [ J ]. China journal of bioengineering, 2016,36(4):1-11 (James c. global Biotechnology/GM cooperative mutation evolution true trend in 2015[ J ]. China Biotechnology 2016,36(4):1-11(in chicken));

[2] triton, Zhangzheng rock, transgenic Soybean research and application progress [ J ]. northwest Agreement, 2016,25(8): 1111-;

[3] bin, hou, yulienhei, research progress of transgenic plant detection technology [ J ]. journal of chinese bioengineering, 2010,30(2): 120-;

[4] liubei, research methods and developments of flanking sequences of foreign Gene Insertion sites [ J ] agriculture and technology, 2012 (4): 97 (Wang X B, Jiang L X,2, Wei L, et al, integration and Insertion Site of EPSPs genes on the Soybean Genome in genetic Modified Glyphosate-resistant Soybean [ J ] ACTA AGRONOMICA SINICA 2010,36(3): 365-;

[5]Fulton T M,Chunwongse J,Tanksley S D.Microprep protocol forextraction of DNA from tomato and other herbaceous plants[J].Plant MolecularBiology Reporter,1995,13(3):207-209。

SEQUENCE LISTING

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Claims (13)

1. A right flank sequence of a transgenic soybean WH8055 transformation event foreign insertion vector, characterized in that the nucleotide sequence is as shown in SEQ ID NO:4, respectively.

2. The use of the right flanking sequence of claim 1 in detecting transgenic soybean, wherein the T-DNA fragment of pCHA L1 is inserted into the transgenic soybean at 43998179 th nucleotide of chromosome 3, wherein the transgenic soybean is WH8055 or transgenic soybean transformed by the T-DNA fragment.

3. The use of claim 2, wherein the right flanking sequence of claim 1 is designed as a PCR primer pair or hybridization probe for the detection of the transgenic soybean.

4. The PCR primer pair designed according to the right flanking sequence of claim 1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO: 6 and SEQ ID NO: shown at 7.

5. A PCR detection kit comprising the primer set according to claim 4 and other reagents.

6. The kit of claim 5, wherein the additional reagents comprise one or more of DNA polymerase, dNTPs, PCR reaction buffer, positive control, and negative control.

7. The kit of claim 6, wherein 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.

8. The kit of claim 6, wherein the DNA polymerase is Taq DNA polymerase.

9. The kit of claim 8, wherein the Taq DNA polymerase is Hot Start Taq DNA polymerase.

10. The kit of claim 5, wherein the additional reagents comprise a pre-mix of PCR reactions, the pre-mix being a mixture of DNA polymerase, dNTPs and PCR reaction buffer.

11. The method for using the primer pair according to claim 4 or the kit according to any one of claims 5 to 10, wherein the method comprises:

(1) performing PCR amplification by using DNA of a soybean sample to be detected as a template and using the primer pair of claim 4 or the primer pair in the kit of any one of claims 5 to 10 and other reagents;

(2) and judging whether a T-DNA fragment of pCHA L1 is inserted at the 43998179 th nucleotide on the No. 3 chromosome of the soybean to be detected or not according to the PCR amplification product.

12. The method according to claim 11, wherein the annealing temperature of the PCR reaction is 49 to 51 ℃ and the number of cycles is 28 to 32.

13. The method of claim 12, wherein the annealing temperature of the PCR reaction is 50 ℃ and the number of cycles is 30.

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