KR101998526B1 - A primer set for detecting active transposon PTE-1 in Chinese cabbage transformants - Google Patents

Abstract

The present invention relates to a primer set for detecting active transposon PTE-1 in Chinese cabbage transformants. The primer set identified in the present invention can be advantageously used to easily and quickly determine the individual whose transposon PTE-1 is activated by the transformation process, using PCR, and furthermore, can be advantageously used to develop a genetically stable transformant.

Description

{A primer set for detecting active transposon PTE-1 in Chinese cabbage transformants (PTE-1), which is a specific activity transcription factor of Chinese cabbage transformants,

The present invention relates to a primer set for detecting a transcription factor PTE-1 that is specifically activated in a Chinese cabbage transformant.

Plant tissue culture refers to a technique of using the plant's typical performance to cultivate plant cells or tissue organs in the artificial medium through somatic cell division and cultivate the same offspring as the parent. Plant transgenesis is a technique for introducing a foreign gene or a part thereof into a plant based on plant tissue culture, and it is a molecular genetic technique that has become more and more popular due to the recent development of genomics, which makes it possible to find a useful gene on a large scale. Plant transformation methods include Agrobacterium, particle bombardment, electroporation, microinjection, and the like.

However, many plants that are cultured and transformed on board are exposed to a variety of stress conditions. There have been reports of genetic and phenotypic changes in transgenic plant cultures and many transgenic plants developed by genetic engineering techniques, and studies on the activation conditions of these transgenic factors It is actively proceeding.

Transposable elements (TEs) are DNA fragments that can move from one place to another in a genome. They are transferred from one chromosome to another by transposition, so they have genetic diversity. It is an important factor to cause. Transcription factors are also called transposons, which range in length from hundreds to thousands of nucleotides. Each transfection factor has a transposase gene capable of cleaving and ligating the DNA necessary for its movement, and each transfection factor has a DNA sequence that is recognized by the transgene.

Transition factors are classified into class I (class I) and class II (class II). Transcription factors belonging to class I are replicated and translocated in the chromosome through RNA intermediates, which are present in the genome at a very high copy number. Transition factors belonging to class I include retrotransposon, short interspersed nuclear element (SINE) and long interspersed nuclear element (LINE). On the other hand, transcription factors belonging to class II translocate in the chromosome through the DNA mediator and exist as relatively low number of copies in the genome. Transition factors belonging to class II are characterized by having terminal inverted repeats (TIRs) regardless of their ability to transfer. Transition factors belonging to class II include Ac / Ds, En / Spm and MITE.

The transition mechanism of MITE (Miniature Inverted Repeat Transposable Elements) has not yet been clarified, but it is classified as class II as a transposition factor consisting of a relatively short DNA sequence of 800 bp or less. MITE has a TIR at both ends and is classified according to its structure. Unlike the transcription factors belonging to general class II, MITE is presumed to exist as a large number of copies on the plant genome, and thus the dislocation of MITE is expected to affect plant genome structure and phenotype expression. P-MITE (plantMITE, http://pmite.hzau.edu.cn/django/mite) is a database that provides information on MITE of 3,527 families obtained from 41 plant genomes including Chinese cabbage, Has made a lot of contributions to research.

Various methods have been reported as methods for detecting polymorphism of the gene at the level of nucleic acid (DNA). Examples of the polymorphism detection markers include RAPD (randomly amplified polymorphic DNAs) marker based on polymerase chain reaction (PCR) amplified fragment length polymorphic DNA markers, microsatellite markers, and simple sequence repeat (SSR) markers.

Therefore, there is a need to develop a molecular marker that can select the activity of the metastatic factor and predict the somatic nutritional status by the metastatic factor. The development of such molecular markers is expected to contribute greatly to the early screening and screening of somatic cell nutrition mutants by tissue culture and transformation.

On the other hand, Chinese cabbage is one of the four major vegetables of Korea as the main ingredient of kimchi and is included in the lacquer ( Brassica ). Rice ( Brassica ) is an important crop in foreign countries because it includes crops with excellent industrial economics such as oilseed rape, mustard.

As a result, the present inventors have made intensive researches to find a transcription factor that specifically changes activity by transformation. As a result, the transcription factor that changes the activity in the Chinese cabbage transformant was identified, and the excised transcription factor was PCR- The present inventors have completed the present invention by confirming that a primer capable of detecting the target gene can be used for assaying the activity of the transfer factor.

(0001) Korea Patent No. KR 10-0753676

It is an object of the present invention to provide a primer set for a Chinese cabbage transfer factor PTE-1 activity assay, which comprises a primer comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

Yet another object of the present invention is to provide a method for assaying the cabbage transfer factor PTE-1 activity.

It is still another object of the present invention to provide a kit for assaying the Chinese cabbage transfer factor PTE-1 comprising the primer set.

The inventors of the present invention have made intensive studies to find a transcription factor that specifically changes the activity of a transformant. As a result, it has been found that the genome of the Chinese cabbage transformant has a high frequency of polymorphism within or adjacent to the transcription factor having the sequence of SEQ ID NO: It is found out that it is occurring.

Accordingly, the present invention relates to a method for producing a marker for assaying the activity of a transfer agent in a plant genome, and comprises the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 using the nucleotide sequence of a transfer factor and / 1 < / RTI > activity test of a Chinese cabbage transfer factor < RTI ID = 0.0 > PTE-1 < / RTI >

In one embodiment of the present invention, the cabbage transfer factor PTE-1 comprises the nucleotide sequence of SEQ ID NO: 3.

Further, the present invention provides a method for producing a Chinese cabbage, comprising: separating a genomic DNA from a Chinese cabbage sample;

(PCR) using a genomic DNA as a template and a primer set for the assay of the Chinese cabbage transfer factor PTE-1 according to claim 1; And

And analyzing the amplification product obtained by the polymerase chain reaction. The present invention also provides a method for assaying the activity of a Chinese cabb mutation factor PTE-1.

In one embodiment of the present invention, if the obtained amplification product is 856 bp, the transfer factor PTE-1 is a deactivated cabbage, and when the amplification product is 580 bp, the transfer factor PTE-1 is activated cabbage.

The PTE-1 activated cabbage is a transformed cabbage.

The present invention provides a primer set for discriminating the activity of PTE-1 in Chinese cabbage and a discrimination method using the primer set, thereby discriminating individuals in which PTE-1 is activated by the tissue culture and transformation, And can be usefully used for developing a stable transformant.

1 is a schematic diagram of a method for analyzing the transfer factor of the present invention and a method for preparing a primer set that specifically discriminates the activity of PTE-1.
Fig. 2 is a graph showing the results obtained by PCR using the primer set for the assay of the transcription factor PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2 and showing that the gene fragment amplified from Chinese cabbage CT001 and Chinese cabbage transformants, Here is the photo:
M: 100bp size marker, CTOO1, IGA6 cabbage.
FIG. 3 is a graph showing the results of PCR of the Chinese cabbage strain CT001, which has not been subjected to tissue culture by PCR using the primer set for the activity assay of the PTE-1, and the base of the fragment of the gene amplified from the Chinese cabbage transformant The results are shown in Fig.
FIG. 4 shows the results of electrophoresis of the primers set for the activity test of the transduction factor PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2 by applying to the Chinese cabbage CT001 individuals not subjected to tissue culture,
M: 100bp size marker, 1-20 Chinese cabbage CT001 individuals not undergoing tissue culture.
FIG. 5 shows the results of applying the primer set for the activity test of the transfer factor PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2 to various cultured Chinese cabbage transformants (BTTP, CO, PPi) The results are as follows:
M: 100 bp size marker, N: CT001 (control group), 1-20 Chinese cabbage transformants.
FIG. 6 shows the results of electrophoresis of the primers set forth in SEQ ID NO: 5 and SEQ ID NO: 6 by applying them to various cabbage transformants (BTTP, CO, PPi)
M: 100 bp size marker, N: CT001 (control group), 1-15 Chinese cabbage transformant.
FIG. 7 shows the results of electrophoresis of the primers set forth in SEQ ID NO: 7 and SEQ ID NO: 8 by applying them to various Chinese cabbage transformants (BTTP, CO, PPi)
M: 100 bp size marker, N: CT001 (control group), 1-15 Chinese cabbage transformant.
FIG. 8 shows the results of electrophoresis of the primers set forth in SEQ ID NO: 9 and SEQ ID NO: 10 by applying them to various cabbage transformants (BTTP, CO, PPi)
M: 100 bp size marker, N: CT001 (control group), 1-15 Chinese cabbage transformant.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited by the following examples.

Example  1: activated in cabbage transformants Metastatic factor  quest

After comparing the activities of MITE based on the "P-MITE Database" and the resequencing data of transformants, we selected the places where MITE was judged to be activated and then prepared a primer set (Fig. 1).

Specifically, referring to P-MITE (plant MITE database), Brassica Sequence information of MITE superfamily DTA, DTC, DTH, DTC and DTT group of rapa (Chiffu strain) was obtained. Using BLAST, the sequence of MITE was mapped to the genome of CT001 strain of Chinese cabbage used for transformant, and the location of MITE on CT001 genome was confirmed. After that, dielectric reanalysis data of the Chinese cabbage transgenic T1 generation (IGA6, IGA7) and T3 generation (IGA634, IGA743) were mapped and aligned with the CT001 genome to analyze the activity of MITE. At this time, the reads are divided into three regions and mapped to both adjacent regions of the MITE region and the corresponding MITE. If both MITE and MITE read areas are mapped, it is determined that MITE is inactivated. If the MITE area is mapped to a depth of at least 3 reads while both adjacent areas of MITE are sufficiently mapped, MITE is activated Respectively. The data were analyzed by using the genomic reanalysis data of Inbred line CT001, Transgenic T1 generation (IGA6, IGA7) and T3 generation (IGA634, IGA743).

Example  2: primer  Set production

Analysis showed that MITE, which showed a significant difference in MITE activation among the T1 generation of IGA6, was selected. The selected MITE was analyzed as MITE belonging to DTH_Brr40 family located on chromosome 3 of CT001, and was named PTE-1. After confirming the nucleotide sequences of both adjacent MITE and MITE regions, four pairs of primers consisting of 20 mers at a distance of 200-300 bp in the MITE region were prepared and the sequence thereof is shown in Table 1 below. When PCR was performed using the PTE-1 F and PTE-1 R primers, the product was 856 bp when MITE was not active and 580 bp when it was active. When PCR was carried out using TE_296_F and TE_296_R primers, 719 bp of active MITE and 423 bp of active MITE were detected. When PCR was performed with TE_251_F and TE_251_R primers, 614 bp of MITE was not active and 614 bp The PCR product of the 363 bp product, TE_365_F and TE_365_R primers, was expected to have 882 bp in the absence of MITE and 517 bp in the active.

primer Base sequence MITE inactive MITE activity PTE-1 F (SEQ ID NO: 1) 5'-CGGACCATTAGTCTCCTATT-3 ' 856 bp 580 bp PTE-1 R (SEQ ID NO: 2) 5'-GGAAGGGAAGTCTACGAG-3'-3 ' TE_296_F (SEQ ID NO: 5) 5'-GCAGATTAGGTCAACTCGG-3 ' 719 bp 423 bp TE_296_R (SEQ ID NO: 6) 5'-CCCTTCTCCAACTTAGCAAT-3 ' TE_251_F (SEQ ID NO: 7) 5'-CGACAGGATTGATGGCGAT-3 ' 614 bp 363 bp TE_251_R (SEQ ID NO: 8) 5'-GCACCGTTAAACACACAGC-3 ' TE_365_F (SEQ ID NO: 9) 5'-CCTGAAGTAACATATAATGAAGGTC-3 ' 882 bp 517 bp TE_365_R (SEQ ID NO: 10) 5'-CTCTTTCATGGTACATGGGTATCG-3 '

Example  3: primer  Set black

The primer set for the assay of the PTE-1, which is shown in SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention, was used to confirm whether or not the activity of PTE-1 in the cabbage transformant after the tissue culture process can be determined .

Specifically, a sample of Chinese cabbage CT001 without tissue culture and a Chinese cabbage transformant IGA6 used for transcription factor screening were prepared and a DNA sample was prepared. The DNA of Chinese cabbage was extracted from leaves of each sample using SDS buffer. The leaf of Chinese cabbage ( Brassica rapa ssp. Pekinensis ) was disrupted using liquid nitrogen, 800 μl of SDS buffer solution was added, and the mixture was reacted at 60 ° C. for 30 minutes. 800 μl of PCI solution (Phenol: Chloroform: Isoamyl alcohol 25: 24: 1) was added and mixed, followed by mixing at room temperature for 10 minutes. The mixture was centrifuged at 20,000 rpm for 15 minutes at 13,000 rpm, and the supernatant was transferred to a new tube. 880 μl of ethanol and 40 μl of 3M sodium acetate were added to precipitate the DNA, and the precipitate was washed with 75% ethanol. Then, it was dried so that there was no ethanol, and dissolved in a TE buffer solution (Tris-EDTA buffer).

In order to confirm the activity of PTE-1 in Chinese cabbage CT001 and Chinese cabbage transformant IX6 that had not undergone tissue culture, the extracted DNA was used to express the transcription factor PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2 PCR assays were performed with primer set for activity assays and tag polymerase (Taq polymerase). The PCR program was run for 40 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally a final extension at 72 ° C for 10 minutes After that, the whole reaction was terminated by maintaining the temperature at 4 ° C. The amplified products were separated on 1% agarose gel for 1 hour and observed with a UV illuminator.

In addition, the amplified product from the cabbage was electrophoresed by PCR using a primer set for the assay of the transit factor PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2, and in the case of the non-tissue cultured, 856 bp The corresponding PCR product was confirmed, and a PCR product corresponding to 580 bp was confirmed in the transformant IGA6 (FIG. 2).

Example  4: amplified PCR  Product Metastatic factor  Check for PTE-1 sequence

In order to analyze the nucleotide sequences of the 856 bp and 580 bp PCR products amplified with the primer set for the activity assay of PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2, the PCR product was used for the base sequence analysis vector pGEM-T easy vector to construct a recombinant vector. The nucleotide sequence of the PCR fragment cloned inside the pGEM-T easy vector for nucleotide sequencing was examined using the T7 and SP6 primers close to the insertion site. The presence of the MITE nucleotide sequence was confirmed using the GenBank BLAST program Respectively.

As a result, it was confirmed that the nucleotide sequence of the 856 bp product contained the base sequence of PTE-1, and the base sequence of the 580 bp product did not contain the base sequence of PTE-1. 4 (Fig. 3).

Through these results, a primer set for the activity test of the PTE-1 having the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 which can specifically confirm the activity of PTE-1 in Chinese cabbage was prepared by the method of the present invention Respectively.

Example  5: primer  Of polymorphism in Chinese cabbage transformants

A procedure was performed to confirm whether or not the activity of PTE-1 can be discriminated even in a cabbage transformant using a primer set for assaying the activity of PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2.

Specifically, gDNA from the Chinese cabbage CT001 individuals not subjected to tissue culture was isolated by the method described in Example 3, and using primer sets for assaying the activity of the transfer factor PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2 PCR was performed.

As a result, it was confirmed that PTE-1 was not activated in the whole of the Chinese cabbage without tissue culture (Fig. 4).

In addition, gDNA was isolated from the various types of Chinese cabbage transformants such as BTTP, CO, and PPi by the method described in Example 3, and the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2, the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 , A primer set of SEQ ID NO: 7, and a primer set of SEQ ID NO: 9, and a primer set for the activity assay of a transfer factor PTE-1 represented by a set of primers of SEQ ID NO: 10.

As a result, only the PCR primer set for the activity assay of the PTE-1, which is shown in SEQ ID NO: 1 and SEQ ID NO: 2, was used to determine the transcription factor activity in BTTP, CO, PPi based Chinese cabbage transformants (Fig. 5).

On the other hand, as a result of performing PCR using primers set forth in SEQ ID NO: 5 and SEQ ID NO: 6, it was not possible to specifically discriminate the transcription factor activity in BTTP, CO, PPi based Chinese cabbage transformants (FIG. 6).

As a result of performing PCR using primers set forth in SEQ ID NO: 7 and SEQ ID NO: 8, it was not possible to specifically discriminate the transcription factor activity in BTTP, CO, PPi-based Chinese cabbage transformants (FIG. 7).

In addition, as a result of performing PCR using primers set forth in SEQ ID NO: 9 and SEQ ID NO: 10, it was not possible to specifically discriminate the transcription factor activity in BTTP, CO, PPi-based Chinese cabbage transformants (FIG. 8).

Therefore, only the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can analyze whether or not the transplant base sequence is contained in the Chinese cabbage transformant. Therefore, the primer set of SEQ ID NO: 1 and SEQ ID NO: , It was confirmed that the activity of PTE-1, which is frequently activated in transformed cells that have undergone tissue culture or tissue culture, can be assayed.

A primer set for the activity assay of the transcription factor PTE-1 shown in SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention was used to early select a somatic cell nutritional mutant by tissue culture and transformation to obtain a transformant having a stable genome It is expected that it will be useful for developing.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> University-Industry Cooperation Group of Kyung Hee University <120> A primer set for detecting active transposon PTE-1 in Chinese          cabbage transformants <130> PN1811-465 <150> KR 10-2018-0126666 <151> 2018-10-23 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTE-1 Forward primer <400> 1 cggaccatta gtctcctatt 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PTE-1 Reverse primer <400> 2 ggaagggaag tctacgag 18 <210> 3 <211> 276 <212> DNA <213> Unknown <220> <223> PTE-1 <400> 3 gggggtgatt ggtagctgct gtaggtgctg tccacagccc tatttatttt cacagcacca 60 aattcagagc aatcaagctt taaatttttt tttgaaacca catctcttaa aatatcctac 120 agctgtacaa gtgctctgca gagccaaata tccaaagcaa ttttttagtg cttcaataaa 180 attctacagc aataaaatct aaagccacag caaaaagtct aaagatttgt ttctacagca 240 aaatatttaa agctacagcg attaccaatc ggaccc 276 <210> 4 <211> 856 <212> DNA <213> Brassica rapa <400> 4 cggaccatta gtctcctatt ccccaacttc cataatctcc ctccttcgca tcttcaccct 60 ccattttcaa cctatatcct tacgatctcg acacctgacg tttttgttga ttcacacacg 120 tatgtcacaa ttctcaattc tcatattttc acaattccca atattttcac agttctctaa 180 atattagggg gtgattggta gctgctgtag gtgctgtcca cagccctatt tattttcaca 240 gcaccaaatt cagagcaatc aagctttaaa tttttttttg aaaccacatc tcttaaaata 300 tcctacagct gtacaagtgc tctgcagagc caaatatcca aagcaatttt ttagtgcttc 360 aataaaattc tacagcaata aaatctaaag ccacagcaaa aagtctaaag atttgtttct 420 acagcaaaat atttaaagct acagcgatta ccaatcggac ccttagtccc acgaatgtag 480 cttttgatgc attaatacaa agtcttaata ttcttttaaa catctttact catcgattat 540 tctctgaaaa atgtgtcctc tgtttctccc ttcttcgtct tcctcctctg ctctgtttct 600 acttcttctc ctcctcctca ctctccaaac cctaacctcc atcactctct cacagcccga 660 agctctccct tcactagaaa agtgcggaaa cttctcaatc tctttccctt tccacctctc 720 ttcctcctcc tcctcctccc ccgttgcttt tcgacttact tgcgcaaact catcaactct 780 gtttctccac ataaaccacc aaacctaccg agtcatcgag ttcttcacag acggtctcct 840 cgtagacttc ccttcc 856 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TE_296_F <400> 5 gcagattagg tcaactcgg 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TE_296_R <400> 6 cccttctcca acttagcaat 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TE_251_F <400> 7 cgacaggatt gatggcgat 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TE_251_R <400> 8 gcaccgttaa acacacagc 19 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TE_365_F <400> 9 cctgaagtaa catataatga aggtc 25 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TE_365_R <400> 10 ctctttcatg gtacatgggt atcg 24

Claims (6)

A primer set for a transposable element PTE-1 activity assay comprising primers comprising the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. 2. The primer set according to claim 1, wherein the cabbage transfer factor PTE-1 comprises the nucleotide sequence of SEQ ID NO: 3. Separating the genomic DNA from the Chinese cabbage sample;
(PCR) using a genomic DNA as a template and a primer set for the assay of the Chinese cabbage transfer factor PTE-1 according to claim 1; And
And analyzing the amplification product obtained by the polymerase chain reaction. 4. The method according to claim 3, wherein the amplification product is 856 bp, wherein the transcription factor PTE-1 is a deactivated Chinese cabbage, and when the amplification product is 580 bp, the transcription factor PTE-1 is activated cabbage. . 5. The method according to claim 4, wherein the PTE-1-activated Chinese cabbage is a transformed Chinese cabbage. A kit for activating a Chinese cabbage transfer factor PTE-1 comprising the primer set of claim 1.

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