KR20160076120A - Primer, Probe for Detecting a Genetically Modified Resveratrol Synthesis Rice and Detecting Method using the Same - Google Patents

Abstract

The present invention relates to a primer and a probe for inspecting a resveratrol biosynthetic genetically modified rice plant, and a method for inspecting a genetically modified rice plant by using the same. More specifically, the present invention relates to a primer and a probe which are specific to each of endogene of a rice plant and an insertion gene inserted in a genetically modified resveratrol biosynthetic rice plant, and a method for inspecting a genetically modified rice plant through a polymerase chain reaction (PCR) by using a standard plasmid. The primer, the probe, and the standard plasmid according to the present invention may specifically detect a genetically modified resveratrol biosynthetic rice plant. In particular, a primer and probe set according to the present invention, as a system-specific primer, may detect a genetically modified rice plant in a particular system, so the genetically modified rice plant in the corresponding system may be monitored and posterior records thereof may be traced.

Description

TECHNICAL FIELD [0001] The present invention relates to a genetic modified resveratrol biosynthesis rice primer, a probe, and a detection method using the genome modified resveratrol rice.

The present invention relates to a primer, a probe, and a method for assaying a genetically modified rice using the same. More particularly, the present invention relates to a primer, a probe, and a method for assaying a genetically modified rice of a resveratrol biosynthesis gene, The present invention relates to a method for assaying transgenic rice plants through polymerase chain reaction (PCR) using primers, probes and standard plasmids.

Genetically Modified (GM) crops worldwide are growing at a rate of 94 times more than in 1996, when it was first cultivated with 160 million hectares, and 19 crops of 25 GM crops were approved for commercialization. Biotechnology Application Information Service (ISAAA) is reporting [James C., 2012, ISAAA Brief NO. 44]. There are no GM crops approved for commercialization and cultivation in Korea, but useful GM crops are being developed for major crops of economic value such as rice, tomato, potato, bean, pepper, cabbage, and perilla. [ Kor . J. Intl . Agri . , 2010. DEC; 22 (4): 399-405; J Plant Biotechnol . , 2011. Jun; 38: 143-150. In particular, rice, which is an important energy source and used as a stock ( Oryza sativa ) are being developed for biotech crops.

Rice is one of the major agricultural products of Southeast Asian countries, including Korea, and is the most dominant in cultivation area, production and consumption. The development of GM rice is actively carried out in the United States, Japan, China, India and Korea. Rice transformants were obtained from Toriyama et al . [ Nat . Biotechnol . , 1998. 6: 1072-1074], which led to great interest in agricultural biotechnology. So far, a variety of GM rice has been developed worldwide, including LLRICE 62 and related events of herbicide resistance by Bayer Crop Science, Shanyou63 of pest resistance developed by Huazhong University of China, and pollen from the National Institute of Agricultural Science (NIAS) Allergy-resistant 7Crp # 10 GM rice was approved by the regulatory authority [ISAAA Database, http://www.isaaa.org/gmapprovaldatabase/]. In addition, Golden Rice was developed to overcome the problem of vitamin A deficiency in infants and children [ Nat . Biotechnol . , 2005. Mar; 23: 482-487; Science , 2000. Jan; 287 (5451): 303-305], it will soon be possible to supply in developing countries if necessary. The development of GM crops with functionalities such as vitamins and minerals or pharmaceuticals and vaccines opens up the era of GM crops, which are free from primary GM crops that provide pest and herbicide-resistance agricultural benefits and that directly benefit human health have.

In order to commercialize GM crops, it is required to evaluate environmental risk and human stability. In Korea, it is required to promote the commercialization of GM crops in accordance with the Guideline for Assessment of Environmental Risk of Genetically Modified Agricultural Products (Agriculture, Forestry and Livestock Industry, 2002-2) . In its examination guidelines, the contents of Attachment 2, Paragraph 8, specify the molecular biological analysis of the transgene, the detection and expression of the transgene, and the test method for the transgenic agricultural product. In addition, the environmental risk assessment of GM crops is being handled on a case-by-case basis, and since data must be prepared for each item submitted to the screening, , And the development of the assay method is essential for the application of post-monitoring technology to ensure safety due to the unintentional release of GMO (Genetically Modified Organism) [ J Agric Food Chem . , 2004. Jun; 52 (11): 3269-3274; J Korean Soc Appl Biol Chem . , 2012. Jun; 55 (3): 367-375).

As the main test method for GMO, polymerase chain reaction (PCR), which detects the DNA of the transgene, is recommended as an international standard method, and screening for detecting the gene in the T-DNA introduced by the method, Specific, gene-specific and construct-specific methods, and event-specific methods for analysis based on insertional contiguous nucleotide sequences and T-DNA nucleotide sequences in a chromosomal genome inserted with a foreign gene [ J Cereal Sci . , 2006. Mar; 43 (2): 250-257; J Agric Food Chem . , 2005. Mar; 53 (8): 3041-3052; Transgenic Res . , Dec. 2005; 14 (6): 817-831). In particular, the event-specific method detects the specific base region newly inserted by the introduction of the introduced gene, so that it is possible to distinguish between the strains of each transformant, and it is possible to easily grasp a sample of the individual derived by crossing. . So recently it has been recommended by the PCR detection methods of GM crops [Collection of Biosafety Reviews , 2007. 3: 8-41; Eur Food Res Technol . , Dec. 2011; 232 (2): 351-359; J Integr Plant Biol . , 2011. Jul; 53 (7): 539-551).

Recently, domestic rice paddy (Korean Patent Laid-Open No. 10-2014-0088492), in which a resveratrol synthetic gene isolated from peanut was introduced into Dongjin-jin, was developed, and gene introduction and resveratrol synthesis were confirmed. In addition, resveratrol transgenic plants introduced with resveratrol synthetic genes were disclosed to GMO packaging and two strains of 'Iksan 515' and 'Iksan 526' were selected considering the main agricultural characteristics and resveratrol content [RDA, Resveratrol Synthesis Rice field event]. However, the PCR assay method for GM rice introduced with resveratrol gene has not been developed so far. In addition, for the safe post-management of GM crops, there is a need for a method of detecting the relevant gene (s) for introduced genes.

Under these circumstances, the inventors of the present invention have made intensive efforts to develop a method for assaying an introduced gene based on the stability evaluation standard for recently developed genetically modified resveratrol biosynthesized rice, and have introduced it into the 'Iksan 515' strain of genetically modified resveratrol biosynthesis rice Specific primers, probes and standard plasmids for quantitative analysis for the detection of the recombinant genes were prepared, and it was confirmed that specific qualitative and quantitative analysis of the genetically modified rice Iksan 515 strain was possible by the PCR assay using the standard plasmids Thereby completing the present invention.

It is an object of the present invention to provide a system-specific PCR primer, a probe and a standard plasmid capable of assaying transgenic rice for biosynthesizing resveratrol and a test method using the plasmid.

In one aspect of the present invention, the present invention provides a primer set consisting of SEQ ID NO: 6 and SEQ ID NO: 7, and a primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12 As a primer set, a PCR primer set that tests the Iksan 515 line is provided.

The "Iksan 515 strain" of the present invention is a genetically modified resveratrol biosynthetic rice selected in consideration of major agricultural characteristics and resveratrol content, resveratrol synthase 3 ( RS3 ; GenBank Accession No. DQ124938) Was introduced into Dong Jinbin and developed to biosynthesize resveratrol.

In the present invention, the term "resveratrol" is a polyphenolic substance having a strong antioxidative property which is produced when a plant is exposed to a bad environment such as mold or insect pests, and is known to be contained in excessive amounts in grape skin, grape seed and peanut. There have been many studies on the effects of resveratrol on humans, and the effects of anticancer, antiviral, neuroprotection, anti-aging, anti-inflammation, and prolongation of life have been known.

The term "resveratrol synthetic gene" in the present invention means a gene (nucleic acid sequence) encoding an enzyme having a function of synthesizing resveratrol, and may finally include all enzymes having a function of synthesizing resveratrol.

The term "primer" in the present invention means a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a template of a complementary nucleic acid, Lt; RTI ID = 0.0 > nucleic acid < / RTI > The primer can initiate DNA synthesis in the presence of reagents for polymerization reactions (i. E., DNA polymerisation) at appropriate buffer solutions and temperatures.

The primer set of the present invention is a "system-specific primer" based on the sequence of the introduced gene insertion site in the plant genome and the nucleotide sequence in the carrier. When the primer set of the present invention is used, the system can be easily distinguished and it is easy to grasp a model for the later hybrid.

The PCR primer set for testing the 'Iksan 515 strain' of the present invention may have a detection limit of 0.1% or more.

Specifically, in one embodiment of the present invention, the detection specificity of the strain is analyzed by using Ras515P01-1 and Ras515P01-2 primer pairs (SEQ ID NOS: 6 and 7), which are systematic specific primer pairs for Iksan 515 strain , And as a result, PCR-amplified products of the Iksan 515 strain were shown to be capable of systematic detection (Fig. 2B).

Further, as a result of confirming the maximum detection limit (LOD) using the systematic primer pair, it was confirmed that the reaction product was present at a concentration of 0.1% or more (FIG. 3).

The term "PCR (polymerase chain reaction) " in the present invention means a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

The PCR method can be divided into qualitative and quantitative detection methods. Qualitative PCR methods are specifically screening, gene-specific, construct-specific, and system-specific event-specific PCR detection. Screening PCR method is a method of detecting and identifying a gene such as 35S promoter or Nos terminator commonly used from the time of development of a genetically modified crop to the present time, and a gene-specific and structure-specific PCR method is a method of identifying a specific part Is amplified and detected. Gene-specific and structure-specific methods have the disadvantage of being difficult to differentiate between genetically modified strains when transformed carriers with the same modified gene and structure are used (Taverniers et al. 2005; Pan et al. 2006 ). In order to solve these drawbacks, a systematic PCR method has recently been used. In this method, a complementary primer is prepared based on the nucleotide sequence of a site adjacent to a site where a transgene in a plant genome is inserted, , It is troublesome to analyze the adjacent sequences inserted into the plant and to confirm the copy number of the introduced gene. However, since the specific base region of the system to be examined is used, even in a system having the same transfection carrier, Can be detected by specifically distinguishing only the system having the < RTI ID = 0.0 > This is based on the fact that PCR amplification can not be performed on the non-target line by a specific primer because the position of insertion into the genome of the plant is changed even when the same carrier is transformed. Therefore, this method has the advantage that it is easy to grasp the transgenic lines in individuals with polymorphism by postmatching of genetically modified individuals, and to test genetically modified agricultural products relatively stably (Berdal and Holst-Jensen 2001; Pan et al., 2006). Unlike the protein detection method, the qualitative PCR method can reliably detect not only the raw agricultural products but also the processed products because it is based on a technique for specifically amplifying the introduced gene.

Real-time PCR is a method of quantitative analysis of genetically modified agricultural products that is currently in practical use. The real-time PCR method is a principle of measuring the amount of fluorescence by dissociating a fluorescent material from a probe during PCR amplification using a probe having a specific primer and a fluorescent material connected thereto. The real-time PCR method is similar to the qualitative PCR method in that it detects the specific DNA of the introduced gene. However, since the ratio of the detected gene is measured based on the inherent gene of the plant, the amount of the introduced gene can be quantitatively analyzed . Substantially real-time PCR is being used to determine the incorporation rate of genetically modified agricultural products and the quantitative determination of GM variants of processed food products.

In another aspect, the present invention provides a method for assaying a genetically modified resveratrol biosynthetic rice comprising the following steps:

a) separating the genomic DNA of the genetically modified resveratrol biosynthesis rice;

b) amplifying the exogenously introduced gene of the genetically modified resveratrol biosynthesized rice using the genomic DNA of step a) as a template and using the PCR primer set for the gene-modified resveratrol biosynthesis rice assay of claim 1; And

c) Identifying a part of the amplified foreign gene by electrophoresis.

In the step of assaying the transgenic rice, the assay may be to identify a lineage of the gene introduced for genetic modification.

As described above, the primer set of the present invention is "system specific primer ". Thus, the succeeding hybrid can be accurately distinguished from the transgenic lines transformed with the same gene as the transgenic model used for crossing. For example, as described in Example 3 below, resveratrol GM rice (Iksan 515 strain) can be systemically detected using the primer set of the present invention.

In addition, the method for assaying the genetically modified rice comprises the step of isolating genomic DNA from a rice sample. Methods for isolating genomic DNA from the sample can be performed by a method known in the art. For example, the CTAB method may be used, or a wizard prep kit (Promega) may be used. The target sequence can be amplified by performing amplification reaction using the separated genomic DNA as a template and using the primer set of the present invention as a primer. Polymerase chain reaction (PCR) is preferred as a method for amplifying a target nucleic acid. PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase.

Such PCR methods are well known in the art and the amplified target sequence may be labeled with a detectable labeling substance. The labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The primer set used to amplify the target sequence is as described above.

Methods for detecting the amplification products are well known in the art. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.

In another embodiment, the present invention provides a probe represented by SEQ. ID. NO: 13 capable of quantitatively assaying 'Iksan 515 strain', a genetically modified resveratrol biosynthesis rice.

The term "probe" in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or an mRNA. An oligonucleotide, A probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and can be labeled for easier detection.

The probe of the present invention may be a probe in which the 5 'terminal of the probe is labeled with the fluorescent substance FAM and the 3' terminal is labeled with the quencher substance TAMRA and both of the base sequences are labeled.

In addition, the probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.).

In another aspect, the present invention provides a pSPS515L standard plasmid capable of quantitatively assaying 'Iksan 515 strain', a genetically modified resveratrol biosynthesis rice.

In the present invention, the term "plasmid" refers to a DNA that can grow independently from a chromosome within a cell of a bacterium and can grow independently, and is not an essential gene for the survival of bacteria. In genetic engineering, plasmid in a bacterium is taken out of the cell, digested with a restriction enzyme, inserted into a desired gene, and then introduced into a bacterium for gene recombination.

In the present invention, the term "standard plasmid" means that the gene for SPS (sucrose-6-phosphate synthase: GenBank Accession No. U33175.1) and the PCR product of each strain-specific gene are linked in the same plasmid, (GMO) standards for GMOs in GMOs and non-GM crops. The pSPS515L standard plasmid can be a reference material for the 'Iksan 515 strain'.

Specifically, in one embodiment of the present invention, the PCR product amplified by the rice intrinsic gene primer pair (RiSPS05-1 and RiSPS05-2; SEQ ID NOs: 8 and 9) and the specific primer pair (5126RasL01-1 and 515RasL01-2; SEQ ID NOS: 11 and 12), and inserted into pGEM-T easy vector to obtain E. coli 5α The pSPS515L standard plasmid for the Iksan 515 strain containing the gene of SEQ ID NO: 14 was prepared (FIG. 4).

The term "inherent gene" in the present invention refers to a gene that exists naturally in the genome of a specific crop and exists as a single copy in the whole genome, and can be used as a positive control for informing the presence of a specific crop in gene analysis of a specific crop Specific gene that does not exist in other relatives and can be used as a negative control.

In the present invention, it is preferable to use the SPS gene as an internal gene, though it is not limited thereto.

In yet another aspect, the present invention provides a method for quantitative analysis of exogenous transgene incorporated in transgenic rice comprising the following steps:

a) preparing a standard plasmid according to claim 4 by diluting the standard plasmid by concentration;

b) performing real-time PCR using the standard plasmid of claim 4 diluted by the concentration and the genomic DNA of the genetically modified rice as a template, using a PCR primer set and a probe for assay specific for the exogenously introduced gene of transgenic rice; And

c) calculating the amount (%) of the foreign introduced gene by converting the amount of the amplification product to the genomic DNA of the transgenic rice relative to the standard quantitative curve.

In preparing the standard plasmid by concentration, the standard plasmid may be a pSPS515L standard plasmid for the Iksan 515 strain, and the dilution concentration of the plasmid may range from 2 x 10 ⁷ to 2 x 10 copies , And may be used as a reference material by diluting the solution in a sequential stepwise concentration.

A standard curve for quantification can be provided by real-time PCR analysis using the number of copies diluted in steps of the above-mentioned plasmids as a template and specific primer pairs and probes of the system.

In the present invention, the term "standard curve" refers to a value obtained by measuring the Ct value of the introduced gene Ct / inherent gene (SPS) using the same primer from a reference material known to have a mixing ratio or a genomic copy number, and regression analysis And the linear relationship of the Ct / SPS Ct value of the introduced gene at each concentration obtained.

In the present invention, the term "mixing ratio" means the ratio of the amount of the genetically modified crop to the total amount of the analytical sample (unknown sample), expressed as a percentage (%).

Specifically, in one embodiment of the present invention, 5126RasL01-1 primer (SEQ ID NO: 11), 515RasL01-2 primer (SEQ ID NO: 12) and Fa151Ras (SEQ ID NO: 13) probe set are used as a template for the stepwise diluted pSPS515L standard plasmid Real-time PCR analysis using the standard curve for quantitation of the Chrysoloxan 515 strain was prepared (Fig. 6).

The amount of the amplification product relative to the genomic DNA of the unknown transgenic rice relative to the standard quantitative curve prepared above is calculated to calculate the incorporation rate (%) of the exogenous transgene, whereby the exogenous transgene incorporated into the transgenic rice can be quantitatively analyzed .

The genetically modified rice in which the exogenous transgene is incorporated may be, but not limited to, genetically modified resveratrol biosynthetic rice, more preferably Iksan 515 strain.

In another aspect, the present invention provides a PCR kit for assaying transgenic rice for assaying rice strain of Iksan 515 strain, which is a genetically modified resveratrol biosynthesis rice.

The PCR kit for the genetically modified resveratrol biosynthetic rice assay comprises a primer set consisting of SEQ ID NO: 6 and SEQ ID NO: 7, and one kind of 'Iksan 515 strain selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12 Specific primer set may be included.

Further, a Fa515Ras probe (SEQ ID NO: 13) specific to 'Iksan 515 strain' capable of quantitatively assaying the transgenic rice can be added and a standard plasmid pSPS515L.

The kit may further include a reagent for performing an amplification reaction, and reagents for performing an amplification reaction may be used as long as they are well known in the art. Specifically, the reagent for performing the amplification reaction may be DNA polymerase, dNTPs, and a buffer, but is not necessarily limited thereto.

The primer, probe and standard plasmid according to the present invention can specifically detect genetically modified resveratrol biosynthesized rice. In particular, the primer and probe set of the present invention can be used as a system specific primer, Therefore, it is possible to monitor the genetically modified rice of the relevant line and trace the post history.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a nucleotide sequence (B) adjacent to an introduced gene of a rice transformation vector (A) and an event line (Iksan 515 strain) used for the development of a genetically modified resveratrol biosynthetic rice of the present invention.
FIG. 2 is a diagram showing the results of the detection specificity analysis of the primers against the genetically modified resveratrol biosynthetic rice of the present invention. RasV03-1 and RasV03-2 specific primer pairs (A) for the resveratrol synthase 3 ( RS3 ), a transgene of the present invention, Ras515P01-1 and Ras515P01-2 specific primer pair (B) for the Iksan 515 strain, And the RiSPS04-1 and RiSPS04-2 specific primer pairs (C) for the rice intrinsic gene SPS (sucrose phosphate synthase ( SPS )) were analyzed by performing PCR on the detection specificity of the primer pair to genetically modified resveratrol biosynthetic rice .
FIG. 3 is a graph showing the results of confirming the limit of detection (LOD) of the genetically modified resveratrol biosynthesis rice ginseng, Iksan 515 strain of the present invention.
4 is a schematic diagram showing a schematic diagram of a standard plasmid prepared for the quantitative assay of the genetically modified resveratrol biosynthesis rice, Iksan 515 strain of the present invention.
5 is a diagram showing the nucleotide sequence of a target DNA fragment of a standard plasmid of Iksan 515 strain for quantitative determination of the genetically modified resveratrol biosynthetic rice of the present invention.
6 is a diagram showing a real time PCR quantitative analysis and a standard calibration curve for the genetically modified resveratrol biosynthesis rice Iksan 515 strain of the present invention. Real-time PCR was performed using the RiSPS05-1 and RiSPS05-2 primers and the FaSPS05 probe (A) specific to the rice intrinsic gene SPS, the 5126RasL01-1 and 515RasL01-2 primers specific to the Iksan 515 strain, and the Fa515Ras probe (B) And analyzed quantitatively.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Plant material and DNA  extraction

In order to test the genetically modified resveratrol biosynthetic transgenic rice of the present invention, DNA of the genetically modified rice was extracted and subjected to polymerase chain reaction (PCR) analysis.

Example  1-1: Plant material

The genetically modified resveratrol biosynthetic rice of the present invention was developed by the GM Rice Rice Development Administration which resveratrol synthase 3 ( RS3 ; GenBank Accession No. DQ124938) was transformed into rice and overexpressed in the plant to increase the biosynthesis of resveratrol , like non-GM Dongjinbyeol, Oryza sativa L., Japnica species. The Iksan 515 system was selected as an event of resveratrol biosynthesis GM rice in consideration of resveratrol content.

Non-GM dongjin rice was used as a control plant and non-GM crops such as corn, rapeseed, soybean, potato, Chinese cabbage, tomato, cotton and cucumber were used as a control for PCR specificity.

Example  1-2: DNA  extraction

For genomic DNA extraction of GM rice, non-GM rice and 8 non-GM crops, DNA was extracted using Quiagen's DNA extraction kit. More specifically, 100 mg of a powder sample of rice and each control plant was taken and DNA was extracted according to the method of KITT and the reagent contained in the DNeasy Plant Mini Kit of Quiagen. The extracted DNA was used as template DNA for assaying GM rice. To determine the GM rice, the concentration of DNA extracted by the above method was adjusted to 50 ng / μl and used for polymerase chain reaction (PCR) Respectively.

Example  2: transgenic transformation Resveratrol  Biosynthetic rice detection primer ( primer Production

For the specific detection of the 'Iksan 515' selection system for genetically modified resveratrol biosynthetic rice, an event-specific primer was constructed based on the inserted nucleotide sequence (SEQ ID NO: 1) of the introduced gene and the event system 1 and Table 1).

Specifically, the SPS gene, which is an inherent gene of rice, exists as a single copy gene in the rice chromosome and can be distinguished from other crops when it is detected in rice. Therefore, it is used as an internal gene of PCR assay [ J Agric Food Chem . , 2004. Jun; 52 (11): 3372-3377]. Therefore, in order to identify rice crops, the detection specific primer pair of the rice embryo gene is a DNA sequence of a sucrose-6-phosphate synthase (SPS) gene (GenBank Accession No. U33175.1), a pair of primers RiSPS04-1 and RiSPS04-2 (SEQ ID NOS: 2 and 3) designed to obtain an amplification product of 115 bp was used [Korean Patent Laid-Open No. 10-2014-0066366; Korean J. Intl . Agri . , 2013. 25 (3): 298-306).

In order to obtain a specific assay for resveratrol biosynthesis rice, RasV03-1 and RasV03-1 were used to obtain an 81 bp amplification product based on the nucleotide sequence of resveratrol synthase 3 ( RS3 ; GenBank Accession No. DQ124938) RasV03-2 (SEQ ID NOS: 4 and 5) primer pairs were selected, and Ras515P01-1 and Ras515P01-1 having a 209 bp amplification product as a specific detection primer pair for the Iksan 515 strain were selected based on the adjacent nucleotide sequence inserted in the genome, 2 (SEQ ID NOS: 6 and 7) primer pairs were prepared.

Example  3: Qualitative PCR  Genetic modification through analysis Resveratrol  For biosynthetic rice primer  Evaluation of detection specificity

In order to detect the specificity of detection of the specific primer pairs prepared in Example 2 for the genetically modified resveratrol biosynthetic rice, based on the transgene and the adjacent nucleotide sequences inserted for the detection of the genetically modified resveratrol biosynthetic rice developed in Korea, PCR was performed on biosynthetic GM rice (Iksan 515 strain), non-GM Dong Jin-on rice, and various non-GM crops.

Specifically, the polymerase chain reaction (PCR) analysis was performed using Multiplex PCR PreMix (including Hotstart Top DNA Polymerase, Reaction buffer, and dNTPs; Bioneer, Korea) , Forward and reverse primers of 10 [mu] M and 50 ng / [mu] l of template DNA were added to perform PCR. The PCR was performed at 95 ° C for 10 minutes, followed by 30 cycles of 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute. The final reaction was performed at 72 ° C for 5 minutes DNA amplification reaction was performed in TProfessional Thermocycler (Biometra, Germany). The amplified PCR product was developed on a 2% agarose gel using an electrophoresis apparatus and the band of the reaction product developed under UV was confirmed.

As a result, as shown in Fig. 2, pairs of RasV03-1 and RasV03-2 primers (SEQ ID NOS: 4 and 5) (A in Fig. 2), which are gene specific primer pairs for the resveratrol biosynthesis gene ( RS3 ) (SEQ ID NOS: 6 and 7) (FIG. 2B), which are the system specific primer pairs for the Iksan 515 strain (2 lane) prepared based on the insertion site of the rice chromosomal DNA, Detection specificity. We used non-GM dongjin rice (1 lane) as a control plant and various non-GM crops (4 lane: corn, 5 lane: rapeseed, 6 lane: beans, 7 lane: potato, 8 lane: Tomato, 10 lane: cotton and 11 lane: cucumber).

In addition, PCR-amplified products were not shown in crops other than rice even in the RiSPS04-1 and RiSPS04-2 primer pairs (SEQ ID NOS: 2 and 3), which are specific primer pairs for the intrinsic gene ( SPS ) (FIG.

Through these results, it was confirmed that the genetic modified resveratrol biosynthesized rice plant Iksan 515 strain could be detected using the test primer pairs prepared in the present invention. The primer pairs were used for the environmental risk assessment, It can be applied to post-monitoring.

Example  4: strain specific Primer  Detection Limits of Genetically Modified Event Systems Using LOD ) Measure

In the genetically modified organism (GMO) test, there is a difficulty in detection due to the unintentional amount of incorporation and the presence of trace amounts of DNA during processing. Therefore, for the application of the actual assay method, limit of detection (LOD) Verification is very important. Thus, the maximum detection limit of the detection method using the Iksan 515 strain specific primer pair prepared in Example 2 was confirmed by carrying out duplex-PCR on the DNA concentration of GMO using the rice embedding gene and the event specific primer.

Specifically, in order to perform Duplex-PCR, DNA sample concentration of genetically modified resveratrol biosynthesized rice bran, Iksan 515 strain was adjusted to a concentration of 100-0.05%. At this time, the concentration of each DNA sample was adjusted by mixing DNA of non-GM Dongjinbyeong, which is the same parent strain used for the transformation of rice. (RiSPS04-1 and RiSPS04-2; SEQ ID NOs: 2 and 3) and the primer pair (Ras515P01-1 and Ras515P01-2) of Iksan 515 strain prepared in Example 2, SEQ ID NO: 6 and 7), duplex-PCR was performed according to the PCR method of Example 3. [

As a result, as shown in FIG. 3, the detection limit of the Iksan 515 strain was clearly distinguishable on the electrophoresis, and the reaction product was exhibited at a concentration of 0.1% or more.

From these results, it was confirmed that the detection limit was 0.1% or more, and the reaction limit was 0.1% or more in the case of the genetically modified resveratrol biosynthesized rice bran, Iksan 515 strain. To 0.9% of the genetically modified resveratrol biosynthetic rice.

Example  5: Realtime ( real - time ) PCR  For analysis primer ( primer ) And a probe probe Production

For the quantitative analysis of 'Iksan 515', a selection system of genetically modified resveratrol biosynthetic rice, based on the inserted nucleotide sequence (SEQ ID NO: 1) of the rice-inherent gene and the event system (Iksan 515) Primer pairs and probes suitable for real-time PCR analysis showing optimum reaction to the PCR reaction conditions were prepared (FIG. 1 and Table 2).

Specifically, in the production of primers and probes for real-time PCR analysis, as in the case of the event-specific primer preparation for the selection system of the genetically modified resveratrol biosynthetic rice of Example 2 described above, 'Iksan 515' The detection specific primer pairs were RiSPS05-1, which was constructed to obtain 91 bp amplification products based on the DNA sequence of sucrose-6-phosphate synthase (SPS) gene (GenBank Accession No. U33175.1) RiSPS05-2 (SEQ ID NOS: 8 and 9) primer pair and FaSPS05 (SEQ ID NO: 10) probe were used [ Korean J. Intl . Agri . , 2013. 25 (3): 298-306).

For the real-time PCR analysis of resveratrol biosynthesized rice, the detection primer pair and its probes were selected based on the nucleotide sequence adjacent to the introduced transgene in the genome that can be specifically detected for the Iksan 515 strain.

Primer pairs (5126 LasL01-1 and 515 LasL01-2; SEQ ID NOs: 11 and 12) and probes (Fa515Ras; SEQ ID NO: 13) for detection of the Iksan 515 strain were constructed so that a 101 bp amplification product was formed.

Example  6: Realtime ( real - time ) PCR  Standard plasmid preparation and detection sensitivity evaluation for analysis

Example  6-1: Generation of standard plasmids for transgenic event system

For the quantitative analysis of genetically modified resveratrol biosynthesis rice by real-time PCR, a plasmid containing a PCR-amplified base fragment as a primer for the Iksan 515 strain prepared in Example 5 was used as a standard material, A standard plasmid for quantitative analysis of strains was constructed.

Since the plasmid for quantitative analysis has the PCR product of the inherent gene for each plant and the gene specific transgene in the same plasmid, it is possible to quantitatively calculate the GMO concentration of the sample easily without GMO standard of each GM line and non-GM crop sample can do. In addition, it is possible to always obtain a uniform and constant plasmid by culturing Escherichia coli, thereby reducing the error of quantitative analysis [ J AOAC Int . , 2002. 85 (5): 1077-1089; Transgenic Res . , Dec. 2005; 14 (6): 817-831).

Specifically, for quantitative real-time PCR analysis of rice plants of Iksan 515 strain, the plasmid was prepared using the rice genetic primer pair (RiSPS05-1 and RiSPS05-2; SEQ ID NOs: 8 and 9) A PCR amplification product was obtained using a specific primer pair (5126RasL01-1 and 515RasL01-2; SEQ ID NOs: 11 and 12) for the event specific primer pair, Iksan 515 strain, and the recognition site of the restriction enzyme Sma I (GCCCGGGC ) Were ligated to each other in the form of SPS :: 515 using the primers given thereto, and a DNA fragment of the linked inherent gene and the transgene was inserted into a pGEM-T easy vector (Promega, USA) Vector. Then, the prepared vector was transformed into E. coli 5α To construct a standard plasmid of pSPS515L against the Iksan 515 strain (Fig. 4). After the standard plasmid was constructed, the desired DNA fragment base sequence of the standard plasmid of Iksan 515 strain was analyzed. As shown in Fig. 5, the detection site of pSPS515L against the Iksan 515 strain was 204 bp. For detection of the Iksan 515 strain, a large amount of plasmid DNA for the Iksan 515 strain, which was produced by culturing the transformed E. coli strain 5α, was extracted and used as a standard material for quantitative PCR analysis.

Example  6-2: Detection sensitivity evaluation of standard plasmid

To confirm the accuracy and precision of the use of the standard plasmid prepared in Example 6-1 as a reference material for real-time PCR analysis, the concentration of the copy number of the standard plasmid for the Iksan 515 strain extracted from the culture was measured Quantitative real-time PCR was performed using a reference material diluted to a stepwise concentration in the range of 2 × 10 ⁷ to 2 × 10 copies. At this time, the control was performed by adding ColE1 / TE DNA (Nippon Gene Co., Japan) as a negative control (NTC, no template control) without sample DNA.

Specifically, using the standard plasmid for the Iksan 515 strain prepared in Example 6-1 and the NTC not added with the standard plasmid, diluted for each step with the number of copies in the above range, (FaSPSO; SEQ ID NO: 10, Fa515Ras; SEQ ID NO: 10, Fa515Ras; SEQ ID NOs: 8 and 9; 5126RasL01-1 and 515RasL01-2; SEQ ID NOs: 11 and 12) SEQ ID NO: 13) were added to each well to perform real-time PCR. Real-time PCR was performed at 95 ° C for 10 minutes using a Stratagene Mx3005P system (Agilent Technologies, USA), followed by 40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute.

As a result of the real-time PCR reaction, a standard curve was obtained from the reference material of each inherent gene and event. As shown in FIG. 6, the real-time PCR analysis showed that the calibration curve for the inherent gene SPS used in the analysis of Iksan 515 strain The correlation coefficient (R ² ) and efficiency were 1.0 and 99.9%, respectively, and 0.996 and 104%, respectively, for the insertion site of the transgene, showing the accuracy and accuracy of real - time PCR analysis.

Based on these results, it was confirmed that the standard plasmid of Iksan 515 system exhibited the accuracy of real-time PCR analysis and the reactivity with high sensitivity, and thus could be used as a standard material for real-time PCR analysis.

Thus, using the standard plasmid of the Iksan 515 strain, which is a PCR product of the rice gene-containing gene and the PCR product of the insertion sequence of the introduced gene of the Iksan 515 strain, the genetic modified resveratrol biosynthetic rice strain, Iksan 515 strain Management, and traceability.

Example  7: Genetic modification using standard plasmids Resveratrol  Analysis of incorporation rate of biosynthetic rice

The results of real-time PCR of the standard plasmid of Iksan 515 strain in Example 6-2 were confirmed to be usable as a standard plasmid for quantitative analysis.

In order to analyze the incorporation rate of the genetically modified resveratrol biosynthesized rice using the standard plasmid, real-time PCR was performed in the same manner as the real-time PCR reaction conditions of the standard plasmid of Example 6-2 using genomic DNA of Iksan 515 strain as a template , And then the number of copies and the correction coefficient of the inherent gene and the introduced gene of the Iksan 515 strain according to the calibration standard curve prepared using the real time PCR result of the standard plasmid of the Iksan 515 strain were calculated.

In real-time PCR quantitative analysis using a standard plasmid in the genetically modified crop assay, it is possible to perform accurate analysis by correcting the analysis error between the target gene and the inherent gene of the corresponding GM crop. Therefore, the resveratrol biosynthesis rice plant, Iksan 515 strain The correction factor for the test was calculated according to the following equation.

Correction factor = angle GM  Number of introduced genes per strain / Number of intrinsic genes

As a result, as shown in Table 3, the copy number of the foreign gene introduced into the Iksan 515 strain was 17360, and the correction coefficient was 1.15.

From these results, it can be confirmed that real-time PCR quantitative analysis can be accurately performed using the standard plasmid of Iksan 515 strain system, since the correction coefficient calculated from the Iksan 515 strain system is detected at the same ratio as that of the intrinsic gene.

As a result of the above results, it can be easily and accurately analyzed qualitatively and quantitatively using the PCR primers, probes and standard plasmids for the Iksan 515 system for the genetically modified resveratrol biosynthetic rice bacterium according to the present invention, .

LB: left border
35S-ter: 35S terminator
bar: phosphinothricin acetyltransferase gene
35S: cauliflower mosaic virus (CaMV) 35S promotor
NOS-ter: nopaline synthase terminator
Resveratrol (RS): Resveratrol synthase 3 gene
Ubiqutin promotor: rice ubiquitin promotor
RB: right border

<110> Republic of Korea <120> Primer, Probe for Detecting a Genetically Modified Resveratrol          Synthesis Rice and Detecting Method using the Same <130> P14R12D1644 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 460 <212> DNA <213> Artificial Sequence <220> <223> 515 line-LB flanking sequence <400> 1 ttctaattcc taaaaccaaa atccagtact aaaatccaga tcccccgaat taattcgncg 60 ttaattcagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag caatcttcgg 120 tgtggcgtat gtgtgcgcct gagtgtgtta aggagagttt agccaaagac tgtatccctc 180 ctaatcttta attaataaag ctccccgttt ttcctcgcaa aaaaaaaaag tcgtatcaca 240 tatgtttaaa aaaccaagta cctgtgacat actgacatgg tttgccggcg cggaactgga 300 atctcgccca aattcggggt atgccggttt aaattttaac tttttccttc aaacttctaa 360 attttttgtc acatcgaact ttcctaccca cacaaatttt taacttttcc gttacatggt 420 ttcaatttct tcaaatttct aattttgacg gggatttaaa 460 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RiSPS04-1 <400> 2 aaccgaacgc ctagaagcg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RiSPS04-2 <400> 3 aagggcaaag cgtcacgtac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RasV03-1 <400> 4 actcgaccca tccgtcaaga 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RasV03-2 <400> 5 tccttagcca aacgaaggac a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ras515P01-1 <400> 6 ttaaaaacgt ccgcaatgtg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ras515P01-2 <400> 7 cggcaaacca tgtcagtatg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RiSPS05-1 <400> 8 aagtgttgca ttccccaagc 20 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RiSPS05-2 <400> 9 acatgactgg tgacagacct tca 23 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> FaSPS05 <400> 10 acgtaccgga gatttatcgc ctcacagg 28 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 5126RasL01-1 <400> 11 cgtccgcaat gtgttattaa gttg 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 515RasL01-2 <400> 12 ggagggatac agtctttggc taaa 24 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Fa515Ras <400> 13 cgtatgtgtg cgcctgagtg tgttaagga 29 <210> 14 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> 515 line plasmid DNA fragment sequence <400> 14 aagtgttgca ttccccaagc atcacaagca gtcagacgta ccggagattt atcgcctcac 60 agggaagatg aaggtctgtc accagtcatg tcctgcccgg gcacgtccgc aatgtgttat 120 taagttgtct aagcaatctt cggtgtggcg tatgtgtgcg cctgagtgtg ttaaggagag 180 tttagccaaa gactgtatcc ctcc 204

Claims (9)

A primer set consisting of SEQ ID NO: 6 and SEQ ID NO: 7, and a primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12, as PCR primers for testing the Iksan 515 line set.
a) separating the genomic DNA of the genetically modified resveratrol biosynthesis rice;
b) amplifying the exogenously introduced gene of the genetically modified resveratrol biosynthesized rice using the genomic DNA of step a) as a template and using the PCR primer set for the gene-modified resveratrol biosynthesis rice assay of claim 1; And
c) identifying a part of the amplified foreign gene by electrophoresis.
A probe for Iksan 515 system assay, as shown in SEQ ID NO: 13, which is used in addition to a PCR primer set for assaying the Iksan 515 line of paragraph 1.
PSPS515L standard plasmid containing the gene of SEQ ID NO: 14, which is used for the quantitative assay of exogenous transgene of Iksan 515 strain.
a) preparing a standard plasmid according to claim 4 by diluting the standard plasmid by concentration;
b) performing real-time PCR using the standard plasmid of claim 4 diluted by the concentration and the genomic DNA of the genetically modified rice as a template, using a PCR primer set and a probe for assay specific for the exogenously introduced gene of transgenic rice; And
c) Quantitative analysis of exogenous transgene incorporated in transgenic rice, comprising the step of calculating the amount (%) of the exogenous transgene by converting the amount of amplification product to the genomic DNA of the transgenic rice relative to the standard quantitative curve.
The quantitative analysis method according to claim 5, wherein the transgenic rice of step b) or c) is a resveratrol biosynthesis rice of Iksan 515 strain.
The quantitative analysis method according to claim 5, wherein the assay PCR primer set of step b) is a primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12, and the probe is a probe of SEQ ID NO:
An Iksan 515 line rice assay kit comprising the primer set of claim 1.
[Claim 9] The kit for assaying rice as set forth in claim 8, further comprising, in addition to the primer set, the probe of SEQ ID NO: 13 of claim 3 and the pSPS515L standard plasmid of claim 4.

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