WO2017163546A1 - Genetically-modified crop detection method - Google Patents

Genetically-modified crop detection method Download PDF

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Publication number
WO2017163546A1
WO2017163546A1 PCT/JP2017/000885 JP2017000885W WO2017163546A1 WO 2017163546 A1 WO2017163546 A1 WO 2017163546A1 JP 2017000885 W JP2017000885 W JP 2017000885W WO 2017163546 A1 WO2017163546 A1 WO 2017163546A1
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nucleic acid
base sequence
seq
set forth
detecting
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PCT/JP2017/000885
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French (fr)
Japanese (ja)
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聡 野間
洋介 菊池
真名美 西
聖子 宮武
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株式会社日清製粉グループ本社
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Priority to JP2018507059A priority Critical patent/JP7091237B2/en
Priority to CN201780007479.3A priority patent/CN108474045A/en
Publication of WO2017163546A1 publication Critical patent/WO2017163546A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to a method for detecting a genetically modified crop and a reagent kit for detecting a genetically modified crop.
  • Genetically modified crops that have been developed using genetically modified technology are used as food.
  • many genetically modified crops such as corn, soybeans, rapeseed, cottonseed (cotton), potatoes, etc. are approved for import and sale after a safety review.
  • a labeling system is provided for genetically modified crops and foods made from them, and labeling is mandatory for designated genetically modified crops and processed foods thereof (Non-Patent Documents 1 and 2).
  • genetically modified crops and non-genetically modified crops may be cultivated in nearby places or used in the same processing plant. Therefore, even if a small amount of genetically modified crops and processed foods derived from them are mixed and processed foods using only non-genetically modified crop ingredients and non-genetically modified crops, recombinant DNA derived from genetically modified crops May be detected.
  • Methods for detecting genetically modified crops in test samples such as agricultural products and foods using the same include methods for detecting recombinant DNA by polymerase chain reaction (hereinafter referred to as “PCR”) and enzyme-linked immunosorbent methods. Although there are methods for detecting recombinant proteins, etc., detection by PCR is generally performed, and Non-Patent Document 3 also describes inspection analysis of genetically modified crops using PCR. In order to examine whether or not a genetically modified crop is mixed in a test sample by PCR, a method of amplifying a partial base sequence of recombinant DNA introduced by a genetic recombination technique is common.
  • DREB Dehydration Responsive Element Binding Protein
  • the present invention provides the following (1) to (4).
  • a method for detecting a genetically modified crop by PCR which can specifically amplify a partial sequence of a DREB gene derived from soybean using a nucleic acid in a test sample or a nucleic acid extracted from the test sample as a template.
  • a method for detecting a genetically modified crop (hereinafter referred to as the first method), which comprises a step of amplifying a nucleic acid having a partial sequence of the DREB gene derived from soybean and a step of detecting the amplified nucleic acid, using a simple primer pair.
  • the term invention refers to this invention).
  • a method for detecting a genetically modified crop by PCR which can specifically amplify a partial sequence of the DREB gene derived from cotton using a nucleic acid in a test sample or a nucleic acid extracted from the test sample as a template
  • a method for detecting a genetically modified crop comprising a step of amplifying a nucleic acid having a partial sequence of the DREB gene derived from cotton using a simple primer pair, and a step of detecting the amplified nucleic acid.
  • second a method for detecting a genetically modified crop (hereinafter referred to as “second”), comprising a step of amplifying a nucleic acid having a partial sequence of the DREB gene derived from cotton using a simple primer pair, and a step of detecting the amplified nucleic acid.
  • the term invention refers to this invention).
  • a reagent kit for detecting or quantifying a genetically modified crop introduced with soybean-derived DREB gene by PCR comprising a nucleic acid comprising the base sequence set forth in SEQ ID NO: 1, the sequence set forth in SEQ ID NO: 1 A nucleic acid containing at least 80% of the base sequence in the base sequence, or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 3 and a nucleic acid containing the base sequence shown in SEQ ID NO: 2, SEQ ID NO: 2
  • a reagent kit comprising a primer pair consisting of a nucleic acid comprising at least 80% of the continuous nucleotide sequence in the nucleotide sequence described in the above, or a nucleic acid hybridizing to the nucleic acid comprising the nucleotide sequence described in SEQ ID NO: 4 (Three inventions refer to this invention).
  • a reagent kit for detecting or quantifying a genetically modified crop introduced with a cotton-derived DREB gene by PCR comprising a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 6, A nucleic acid containing at least 80% of the base sequence in the base sequence, or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 8, and a nucleic acid containing the base sequence shown in SEQ ID NO: 7, SEQ ID NO: 7
  • a reagent kit comprising a primer pair consisting of a nucleic acid comprising at least 80% of the base sequence described in the above, or a nucleic acid hybridizing to a nucleic acid comprising the base sequence described in SEQ ID NO: 9
  • the term “four inventions” means this invention).
  • Patent Document 1 also does not describe a method for specifically detecting the DREB gene.
  • an object of the present invention is to provide a method and a reagent kit that can detect a genetically modified crop introduced with a DREB gene with high reliability and practicality.
  • the present invention is a method for detecting a genetically modified crop. That is, a genetically modified crop introduced with recombinant DNA contained in a test sample such as a plant crop, a food material obtained from the crop, or a processed food obtained using the crop or food material is detected. It is a method to do. Examples of plant crops include cereal grains, potatoes, sugar beets, and pineapples.
  • Grain grains include, for example, wheat (wheat, barley, oats, rye, barley, etc.), rice, corn, milo, millet, millet, and other legumes, soybeans, red beans, peanuts, peas, kidney beans, and other beans.
  • the seed of a family plant is mentioned, It is especially preferable that it is wheat.
  • Examples of food materials obtained from the crop include wheat flour, medium flour, whole grain flour, quasi-strong flour, strong flour, durum wheat flour, and rye flour when the crop is a grain other than wheat. , Rice flour and the like.
  • Examples of processed foods obtained using crops and food materials obtained from the crops include tofu and the like when the crop is wheat, bread and noodles, and when the crop is soybean.
  • the recombinant DNA detected in the present invention is the DREB gene.
  • the DREB gene can improve the environmental stress tolerance of crops, and it is expected that the production of genetically modified crops into which the DREB gene has been introduced will be expanded in the future. Therefore, if the DREB gene is used for detection of genetically modified crops in the future, it may be possible to detect genetically modified crops relatively easily.
  • the soybean-derived DREB gene in the test sample is detected by PCR.
  • This detection is carried out by PCR using a primer pair that can specifically amplify a partial sequence of the DREB gene derived from soybean, using the nucleic acid in the sample or the nucleic acid extracted from the sample as a template.
  • a nucleic acid having a partial sequence of the DREB gene is amplified, and the amplified nucleic acid is detected or quantified.
  • the cotton-derived DREB gene in the test sample is detected by PCR.
  • This detection is performed by PCR using a primer pair that can specifically amplify a partial sequence of the DREB gene derived from cotton using a nucleic acid in the test sample or a nucleic acid extracted from the test sample as a template.
  • a nucleic acid having a partial sequence of the DREB gene is amplified, and the amplified nucleic acid is detected or quantified.
  • FIG. 1 shows part of the DNA sequence of each of the DREB gene derived from soybean and the DREB gene derived from cotton.
  • the DNA sequence of the soybean-derived DREB gene is similar to that of the cotton-derived DREB gene.
  • a primer is designed, and in the second invention, it is preferable to design a primer in a DNA sequence portion specific to the cotton-derived DREB gene.
  • preferred primer pairs are the following (1) to (3).
  • the base sequence described in SEQ ID NO: 1 is the base sequence of the region indicated by R1 in FIG. 1 in the entire base sequence of the soybean-derived DREB gene described in SEQ ID NO: 13, and the base sequence described in SEQ ID NO: 2 is It is the base sequence of the complementary strand of the region indicated by R2 in FIG. 1 in the entire base sequence of the soybean-derived DREB gene described in SEQ ID NO: 13.
  • a nucleic acid that hybridizes under stringent conditions to the base sequence described in SEQ ID NO: 3 that is complementary to the base sequence described in SEQ ID NO: 1, and a sequence number that is complementary to the base sequence described in SEQ ID NO: 2 A nucleic acid that hybridizes to the base sequence according to 4 under stringent conditions.
  • the present invention relates to a genetically modified crop in which a soybean-derived or cotton-derived DREB gene is introduced by PCR using a primer pair that specifically and simultaneously amplifies soybean-derived DREB gene and cotton-derived DREB gene. You may detect or quantify.
  • a nucleic acid comprising the base sequence described in SEQ ID NO: 1 in the primer pair of (1) above, comprising at least 80% of the continuous base sequence in the base sequence described in SEQ ID NO: 1 in the primer pair of (2) above.
  • the nucleic acid and the nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 3 that is complementary to the nucleotide sequence of SEQ ID NO: 1 in the primer pair of (3) above are any of them.
  • nucleic acid comprising the base sequence described in SEQ ID NO: 2 in the primer pair of (1) above, and at least 80% continuous in the base sequence described in SEQ ID NO: 2 in the primer pair of (2) above
  • nucleic acid comprising the base sequence described above and the base sequence described in SEQ ID NO: 4 that is complementary to the base sequence described in SEQ ID NO: 2 in the primer pair of (3) above May be used in combination with stringent any one or more of the hybridizing nucleic acids under conditions. Expressions such as claims 2 and 8 of the present application include primer pairs composed of such combinations.
  • preferred primer pairs are the following (4) to (6).
  • the base sequence described in SEQ ID NO: 6 is the base sequence of the region indicated by R6 in FIG. 1 in the entire base sequence of the cotton-derived DREB gene described in SEQ ID NO: 14, and the base sequence described in SEQ ID NO: 7 is It is the base sequence of the complementary strand of the region indicated by R7 in FIG. 1 in the base sequence of the cotton-derived DREB gene described in SEQ ID NO: 14.
  • nucleic acid consisting of a nucleic acid comprising at least 80% continuous base sequence in the base sequence described in SEQ ID NO: 6 and a nucleic acid comprising at least 80% continuous base sequence in the base sequence described in SEQ ID NO: 7.
  • (6) A nucleic acid that hybridizes under stringent conditions to the base sequence described in SEQ ID NO: 8 that is complementary to the base sequence described in SEQ ID NO: 6, and a sequence number that is complementary to the base sequence described in SEQ ID NO: 7
  • a nucleic acid comprising the base sequence described in SEQ ID NO: 6 in the primer pair of (4) above, and at least 80% of the contiguous base sequence in the base sequence described in SEQ ID NO: 6 in the primer pair of (5) above.
  • the nucleic acid and the nucleic acid that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 8 that is complementary to the base sequence set forth in SEQ ID NO: 6 in the primer pair of (6) above are any of them.
  • nucleic acid comprising the base sequence described in SEQ ID NO: 7 in the primer pair of (4) above, and at least 80% continuous in the base sequence described in SEQ ID NO: 7 in the primer pair of (5) above
  • nucleic acid comprising the base sequence described above and the base sequence described in SEQ ID NO: 9 that is complementary to the base sequence described in SEQ ID NO: 7 in the primer pair of (6) above May be used in combination with stringent any one or more of the hybridizing nucleic acids under conditions. Expressions such as claims 5 and 10 of the present application include primer pairs composed of such combinations.
  • Each nucleic acid containing at least 80% of the continuous base sequence in the base sequence described in SEQ ID NO: 1, 3, 6 or 8 is 85 in the base sequence described in SEQ ID NO: 1, 3, 6 or 8. It is preferable that the nucleic acid contains a continuous base sequence of at least 90%, more preferably a nucleic acid containing 90% or more of the continuous base sequence in the base sequence described in SEQ ID NO: 1, 3, 6 or 8.
  • the nucleic acid used as a primer preferably has 10 or more bases, more preferably 12 or more, and still more preferably 15 or more.
  • the primer pair (3) or (6) under stringent conditions, a specific hybrid is formed with the DNA sequence to be amplified, and a non-specific hybrid is not formed with the DNA sequence that is not the amplification target.
  • the pair of nucleic acids constituting the primer pair of (3) or (6) functions as a primer in a state where it is hybridized to a complementary base sequence.
  • Stringent conditions can be readily determined by those skilled in the art based on the length of the DNA, for example. Those skilled in the art can easily determine the appropriate conditions for the selected primer based on the common knowledge of various primer design methods and hybridization conditions known in the art, and empirical rules obtained through commonly used experimental means. Can be found and implemented.
  • the partial sequence shown in SEQ ID NO: 11 (see FIG. 2) of the DNA sequence of the soybean-derived DREB gene shown in FIG.
  • the partial sequence described in SEQ ID NO: 12 (see FIG. 3) of the cotton-derived DREB gene DNA sequence shown in FIG. 1 can be specifically amplified. It can.
  • the term "specific" as used herein means that only the partial sequence in the soybean-derived DREB gene is amplified in the first and third inventions, and in the second and fourth inventions, in the cotton-derived DREB gene. Means that only a partial sequence of is amplified.
  • the partial sequence to be amplified in the first and third inventions is preferably SEQ ID NO: 11, but is not limited thereto, and other regions of the DNA sequence of the DREB gene derived from soybean may be amplified. Good.
  • the partial sequence to be amplified in the second and fourth inventions is also preferably SEQ ID NO: 12, but is not limited thereto, and other regions of the cotton-derived DREB gene may be amplified. Good.
  • the test sample may be subjected to nucleic acid extraction, for example, as it is or after being pulverized, or may be washed, dried, crushed and subjected to nucleic acid extraction.
  • the nucleic acid extracted from the test sample and used for analysis is usually DNA.
  • DNA may be extracted by any known method, but many DNA extraction kits are commercially available and can be extracted using these. For example, using DNeasy Plant Maxi kit (manufactured by QIAGEN), using the method of Kopell et al. (Kopell, E. et al .; DNA may be extracted from the test sample.
  • the extracted DNA is preferably used after calculating its concentration by measuring absorbance and diluting it to a concentration suitable for PCR.
  • PCR can be performed according to a conventional method in consideration of the primer and DNA polymerase to be used.
  • reagents such as a PCR buffer, dNTP, and MgCl 2 may be prepared, or a commercially available PCR kit may be used.
  • PCR conditions can be used, for example, at 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds for 35 cycles, and finally at 72 ° C. for 7 minutes as the end reaction.
  • the Tm value, the length of the region to be amplified, the concentration of the template DNA, and the like can be changed as appropriate.
  • the amplified nucleic acid (hereinafter referred to as “PCR product”) can be detected by any method for identifying a specific DNA fragment. Examples of the identification method include agarose gel electrophoresis, acrylamide gel electrophoresis, capillary electrophoresis, hybridization, and immunological methods. In general, PCR products are identified by electrophoresis patterns. For example, electrophoresis using 0.8% agarose gel containing ethidium bromide may be performed to detect the PCR product as a band.
  • the PCR product can also be identified in the process of PCR amplification by performing real-time PCR in the presence of a primer pair and a nucleic acid probe.
  • a nucleic acid probe used in real-time PCR fluorescent label, radioactive substance label, and biotin-labeled probe can be used.
  • the nucleic acid probe used in the present invention is, for example, modified with FAM using the 5 ′ end of an oligonucleotide having the base sequence described in SEQ ID NO: 5 or SEQ ID NO: 10 as a fluorescent substance, and TAMRA or MGB using the 3 ′ end as a quencher.
  • nucleic acid probe consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 5 or SEQ ID NO: 10, SEQ ID NO: 5 or SEQ ID NO: 10 or these
  • a nucleic acid probe comprising a nucleotide sequence having a homology of 80% or more, more preferably 85% or more, more preferably 90% or more, and a fluorescent label, radioactive substance label or biotin label It can also be used.
  • the third invention and the fourth invention are reagent kits including the primer pairs used in the first invention and the second invention, respectively.
  • the nucleic acid constituting the primer pair can be produced according to a conventional method.
  • the reagent kit may contain a primer pair and another reagent.
  • the reagent kit may include dNTP, MgCl 2 , DNA polymerase (eg, Taq DNA polymerase), buffer (eg, Tris-HCl), glycerol, DMSO, positive control DNA, negative control DNA, distilled water, and the like. Good.
  • the reagent kit may include a fluorescent probe, a radioactive substance label, or a biotin-labeled nucleic acid probe for use in the detection or quantification of the nucleic acid of the amplification product by PCR.
  • Reagents contained in the reagent kit may be individually packed, or may be packed after being mixed. There are no particular limitations on the concentration of each reagent in the reagent kit, and any concentration range can be used as long as PCR according to the present invention can be performed. Further, information such as suitable PCR conditions may be further attached to the reagent kit.
  • Example 1 Detection of soybean-derived and cotton-derived DREB genes by PCR and agarose gel electrophoresis
  • Method PCR was carried out using the DNA extracted from each of the following three test samples as a template and using the following primer pairs. Thereafter, the presence or absence of amplification in each sample was examined by agarose gel electrophoresis.
  • Wheat Domestic wheat Soybean: Dry domestic seeds of commercially available soybeans
  • Cotton Dry seeds of commercially available cotton After each sample was crushed with Multi Beads Shocker (Yasui Kikai Co., Ltd.), DNeasy Plant Maxi kit (QIAGEN) was used. DNA was extracted.
  • Primer pair In order to design primers used for PCR, a gene sequence database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/) was used. Using accession number AF514908 (SEQ ID NO: 13) as the DNA sequence of the DREB gene derived from soybean, a primer pair consisting of a nucleic acid having the base sequence described in SEQ ID NO: 1 and a nucleic acid having the base sequence described in SEQ ID NO: 2 Designed.
  • accession number AF509502 (SEQ ID NO: 14) as the DNA sequence of the DREB gene derived from cotton
  • a primer pair comprising a nucleic acid having the base sequence described in SEQ ID NO: 6 and a nucleic acid having the base sequence described in SEQ ID NO: 7 Designed. Primer synthesis was outsourced to Operon.
  • PCR was performed using DNA extracted from each sample as a template. Primers of SEQ ID NO: 1 and SEQ ID NO: 2 were used for detection of the DREB gene derived from soybean. In addition, primers of SEQ ID NO: 6 and SEQ ID NO: 7 were used for detection of the cotton-derived DREB gene. The PCR reaction mix was adjusted for each of the three test samples.
  • the detection result of the soybean-derived DREB gene is shown in FIG. As shown in FIG. 2, a band of the expected length (91 bp) was detected only for DNA extracted from soybean, and no band was detected for DNA extracted from cotton and wheat. Furthermore, the DNA sequence of the band detected in soybean was the sequence of the amplified region of the DREB gene derived from soybean. The detection result of cotton-derived DREB gene is shown in FIG. As shown in FIG. 3, a band of the expected length (94 bp) was detected only for DNA extracted from cotton, and no band was detected for DNA extracted from soybean and wheat. Furthermore, the DNA sequence of the band detected in cotton was the sequence of the amplified region of the cotton-derived DREB gene. Note that “4.TE” in FIGS. 2 and 3 is a negative control.
  • the DREB genes derived from soybean and cotton can be specifically amplified by PCR using the primer pairs of SEQ ID NOs: 1, 2 and SEQ ID NOs: 6, 7. Therefore, if these primer pairs are used, genetically modified crops other than soybean and cotton other than soybean introduced with soybean-derived and cotton-derived DREB genes can be detected. Moreover, since these primer pairs are not amplified in wheat, it is possible to detect genetically modified wheat introduced with soybean-derived and cotton-derived DREB genes.
  • Example 2 Detection of soybean-derived and cotton-derived DREB genes by real-time PCR
  • Method 2 Real-time PCR was performed using DNA extracted from the same three types of test samples as used in Example 1 as templates and using the following primer pairs and nucleic acid probes.
  • Primer pair For the DREB gene derived from soybean, a primer pair consisting of a nucleic acid having the base sequence described in SEQ ID NOs: 1 and 2 used in Example 1 was used.
  • a primer pair consisting of nucleic acids having the base sequences described in SEQ ID NOs: 6 and 7 used in Example 1 was used.
  • nucleic acid probe For the soybean-derived DREB gene, the nucleic acid probe of SEQ ID NO: 5 was designed in the DNA sequence between the primer pair (SEQ ID NOs: 1, 2). This nucleic acid probe was labeled with FAM at the 5 ′ end and modified with TAMRA using the 3 ′ end as a quencher.
  • a nucleic acid probe of SEQ ID NO: 10 was designed in the DNA sequence between the primer pair (SEQ ID NOs: 6, 7). This nucleic acid probe was labeled with FAM at the 5 ′ end and modified with MGB using the 3 ′ end as a quencher. The synthesis of the nucleic acid probe was outsourced to Applied Biosystems.
  • Real-time PCR was performed using DNA extracted from each sample as a template.
  • a fluorescently labeled nucleic acid probe containing the primers of SEQ ID NO: 1 and SEQ ID NO: 2 and the oligonucleotide of SEQ ID NO: 5 was used.
  • a fluorescently labeled nucleic acid probe containing the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and the oligonucleotide of SEQ ID NO: 10 was used.
  • a real-time PCR reaction mix was prepared for each of the three test samples.
  • the detection result of the soybean-derived DREB gene is shown in FIG. As shown in FIG. 4, the fluorescence intensity increased exponentially only with DNA extracted from soybean, but not with DNA extracted from cotton and wheat. The detection result of cotton-derived DREB gene is shown in FIG. As shown in FIG. 5, the fluorescence intensity increased exponentially only with DNA extracted from cotton, but not with DNA extracted from soybean and wheat.
  • the fluorescently labeled nucleic acid probe containing the primer pair of SEQ ID NO: 1 and 2 and the oligonucleotide of SEQ ID NO: 5 and the fluorescently labeled nucleic acid probe containing the primer pair of SEQ ID NO: 6, 7 and the oligonucleotide of SEQ ID NO: 10 were labeled. It was found that the DREB gene derived from soybean and cotton can be specifically detected by performing real-time PCR using the nucleic acid probe. Therefore, if these primer pairs and nucleic acid probe sets are used, it is possible to detect genetically modified crops other than soybean and cotton other than soybean and cotton-derived DREB genes introduced in real-time PCR, respectively. . Moreover, since these primer pairs and nucleic acid probe sets are not amplified in wheat, it is possible to detect genetically modified wheat introduced with soybean-derived and cotton-derived DREB genes.
  • a genetically modified crop introduced with a DREB gene which is contained in a test sample such as an agricultural product or a food using the same, has high reliability and It can be detected with practicality.

Abstract

The genetically-modified crop detection method is for detecting a genetically-modified crop using a polymerase chain reaction, and comprises: a step for amplifying a nucleic acid having a partial sequence of a soybean-derived DREB gene, by using a nucleic acid in a sample to be tested or a nucleic acid extracted from the sample to be tested as a template, and a primer pair which can be used to specifically amplify the partial sequence of the soybean-derived DREB gene; and a step for detecting or quantifying the amplified nucleic acid. The present invention also provides a reagent kit for detecting or quantifying a genetically-modified crop having an introduced soybean-derived DREB gene using a polymerase chain reaction.

Description

遺伝子組換え作物の検出方法Method for detecting genetically modified crops
　本発明は、遺伝子組換え作物の検出方法及び遺伝子組換え作物検出用の試薬キットに関する。 The present invention relates to a method for detecting a genetically modified crop and a reagent kit for detecting a genetically modified crop.
　遺伝子組換え技術を利用して開発された遺伝子組換え作物が食品として利用されるようになっている。日本においても、トウモロコシ、ダイズ、ナタネ、綿実（ワタ）、ジャガイモ等の多くの遺伝子組換え作物について、安全性審査を経て、輸入や販売が認められている。また、遺伝子組換え作物やそれを原料とする食品について表示制度が設けられており、指定された遺伝子組換え作物及びその加工食品については、その表示が義務づけられている（非特許文献１，２）。
　他方、海外においては、遺伝子組換え作物と非遺伝子組換え作物が近い場所で栽培される、または同じ加工工場内で使用される場合がある。そのため、微量の遺伝子組換え作物及びそれ由来の加工食品が混入し、非遺伝子組換え作物の原料及び非遺伝子組換え作物のみを使用した加工食品であっても遺伝子組換え作物由来の組換えＤＮＡが検出される可能性がある。 Genetically modified crops that have been developed using genetically modified technology are used as food. In Japan as well, many genetically modified crops such as corn, soybeans, rapeseed, cottonseed (cotton), potatoes, etc. are approved for import and sale after a safety review. In addition, a labeling system is provided for genetically modified crops and foods made from them, and labeling is mandatory for designated genetically modified crops and processed foods thereof (Non-Patent Documents 1 and 2). ).
On the other hand, in overseas, genetically modified crops and non-genetically modified crops may be cultivated in nearby places or used in the same processing plant. Therefore, even if a small amount of genetically modified crops and processed foods derived from them are mixed and processed foods using only non-genetically modified crop ingredients and non-genetically modified crops, recombinant DNA derived from genetically modified crops May be detected.
　農産物やそれを使用した食品等の被検試料中の遺伝子組換え作物の検出方法としては、ポリメラーゼ連鎖反応（以下「ＰＣＲ」という）によって組換えＤＮＡを検出する方法や、酵素結合免疫吸着法によって組換え蛋白質を検出する方法等があるが、一般的にＰＣＲによる検出が行われており、非特許文献３にも、ＰＣＲを用いた遺伝子組換え作物の検査分析が記載されている。
　ＰＣＲで被検試料中に遺伝子組換え作物が混入しているか否かを調べるには、遺伝子組換え技術によって導入された組換えＤＮＡの部分塩基配列を増幅する方法が一般的であるが、導入された組換えＤＮＡの種類が不明である場合、標的とする組換えＤＮＡを代えて複数回ＰＣＲを行い、遺伝子組換え作物が含まれていないことを確認する必要がある。しかし、そのような方法は手間がかかり、実用的ではない。
　そのため、農産物やそれを使用した食品等について、遺伝子組換え作物が含まれるか否かに関する表示を適切に行うためには、信頼性と実用性の高い遺伝子組換え作物の検出技術の開発が望まれる。 Methods for detecting genetically modified crops in test samples such as agricultural products and foods using the same include methods for detecting recombinant DNA by polymerase chain reaction (hereinafter referred to as “PCR”) and enzyme-linked immunosorbent methods. Although there are methods for detecting recombinant proteins, etc., detection by PCR is generally performed, and Non-Patent Document 3 also describes inspection analysis of genetically modified crops using PCR.
In order to examine whether or not a genetically modified crop is mixed in a test sample by PCR, a method of amplifying a partial base sequence of recombinant DNA introduced by a genetic recombination technique is common. When the type of the recombinant DNA thus obtained is unknown, it is necessary to perform PCR a plurality of times in place of the target recombinant DNA to confirm that no genetically modified crop is contained. However, such a method is time consuming and impractical.
For this reason, it is desirable to develop a technology for detecting genetically modified crops with high reliability and practicality in order to appropriately indicate whether or not genetically modified crops are included in agricultural products and foods that use them. It is.
　また、遺伝子組換え技術により農産物に導入する外来遺伝子として、環境ストレス耐性に必要な遺伝子群の発現を制御するＤｅｈｙｄｒａｔｉｏｎ　ｒｅｓｐｏｎｓｉｖｅ　ｅｌｅｍｅｎｔ　ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎ（以下「ＤＲＥＢ」という）遺伝子が知られている。このＤＲＥＢ遺伝子を農産物内で過剰発現させることにより、乾燥、塩、低温等の環境ストレスに対する耐性が向上することが報告されている。例えば、特許文献１には、低温等のストレス耐性を形質導入するために有用な新規のポリヌクレオチド（構造遺伝子領域およびその発現制御領域）が開示されている。 Also, as a foreign gene to be introduced into agricultural products by gene recombination technology, a Dehydration Responsive Element Binding Protein (hereinafter referred to as “DREB”) gene that controls the expression of genes required for environmental stress tolerance is known. It has been reported that by overexpressing the DREB gene in agricultural products, resistance to environmental stresses such as drought, salt, and low temperature is improved. For example, Patent Document 1 discloses a novel polynucleotide (structural gene region and its expression control region) useful for transducing stress tolerance such as low temperature.
特開２００２－２２３７５７号公報JP 2002-223757 A
　本発明は、以下の（１）～（４）を提供するものである。
（１）遺伝子組換え作物をＰＣＲによって検出する方法であって、被検試料中の核酸又は前記被検試料から抽出した核酸を鋳型とし、ダイズ由来のＤＲＥＢ遺伝子の部分配列を特異的に増幅可能なプライマーペアを用いて、前記ダイズ由来のＤＲＥＢ遺伝子の部分配列を有する核酸を増幅する工程と、前記増幅された核酸を検出する工程と、を含む遺伝子組換え作物の検出方法（以下、第１発明というときはこの発明をいう）。
（２）遺伝子組換え作物をＰＣＲによって検出する方法であって、被検試料中の核酸又は前記被検試料から抽出した核酸を鋳型とし、ワタ由来のＤＲＥＢ遺伝子の部分配列を特異的に増幅可能なプライマーペアを用いて、前記ワタ由来のＤＲＥＢ遺伝子の部分配列を有する核酸を増幅する工程と、前記増幅された核酸を検出する工程と、を含む遺伝子組換え作物の検出方法（以下、第２発明というときはこの発明をいう）。 The present invention provides the following (1) to (4).
(1) A method for detecting a genetically modified crop by PCR, which can specifically amplify a partial sequence of a DREB gene derived from soybean using a nucleic acid in a test sample or a nucleic acid extracted from the test sample as a template. A method for detecting a genetically modified crop (hereinafter referred to as the first method), which comprises a step of amplifying a nucleic acid having a partial sequence of the DREB gene derived from soybean and a step of detecting the amplified nucleic acid, using a simple primer pair. The term invention refers to this invention).
(2) A method for detecting a genetically modified crop by PCR, which can specifically amplify a partial sequence of the DREB gene derived from cotton using a nucleic acid in a test sample or a nucleic acid extracted from the test sample as a template A method for detecting a genetically modified crop (hereinafter referred to as “second”), comprising a step of amplifying a nucleic acid having a partial sequence of the DREB gene derived from cotton using a simple primer pair, and a step of detecting the amplified nucleic acid. The term invention refers to this invention).
（３）ダイズ由来のＤＲＥＢ遺伝子が導入された遺伝子組換え作物を、ＰＣＲによって検出又は定量するための試薬キットであって、配列番号１に記載の塩基配列を含む核酸、配列番号１に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号３に記載の塩基配列を含む核酸にハイブリダイズする核酸と、配列番号２に記載の塩基配列を含む核酸、配列番号２に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号４に記載の塩基配列を含む核酸にハイブリダイズする核酸とからなるプライマーペアを含む、試薬キット（以下、第３発明というときはこの発明をいう）。
（４）ワタ由来のＤＲＥＢ遺伝子が導入された遺伝子組換え作物を、ＰＣＲによって検出又は定量するための試薬キットであって、配列番号６に記載の塩基配列を含む核酸、配列番号６に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号８に記載の塩基配列を含む核酸にハイブリダイズする核酸と、配列番号７に記載の塩基配列を含む核酸、配列番号７に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号９に記載の塩基配列を含む核酸にハイブリダイズする核酸とからなるプライマーペアを含む、試薬キット（以下、第４発明というときはこの発明をいう）。 (3) A reagent kit for detecting or quantifying a genetically modified crop introduced with soybean-derived DREB gene by PCR, comprising a nucleic acid comprising the base sequence set forth in SEQ ID NO: 1, the sequence set forth in SEQ ID NO: 1 A nucleic acid containing at least 80% of the base sequence in the base sequence, or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 3 and a nucleic acid containing the base sequence shown in SEQ ID NO: 2, SEQ ID NO: 2 A reagent kit comprising a primer pair consisting of a nucleic acid comprising at least 80% of the continuous nucleotide sequence in the nucleotide sequence described in the above, or a nucleic acid hybridizing to the nucleic acid comprising the nucleotide sequence described in SEQ ID NO: 4 (Three inventions refer to this invention).
(4) A reagent kit for detecting or quantifying a genetically modified crop introduced with a cotton-derived DREB gene by PCR, comprising a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 6, A nucleic acid containing at least 80% of the base sequence in the base sequence, or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 8, and a nucleic acid containing the base sequence shown in SEQ ID NO: 7, SEQ ID NO: 7 A reagent kit comprising a primer pair consisting of a nucleic acid comprising at least 80% of the base sequence described in the above, or a nucleic acid hybridizing to a nucleic acid comprising the base sequence described in SEQ ID NO: 9 The term “four inventions” means this invention).
ダイズ由来のＤＲＥＢ遺伝子及びワタ由来のＤＲＥＢ遺伝子それぞれのＤＮＡ配列の一部を並べた図である。It is the figure which arranged a part of DNA sequence of each of the DREB gene derived from soybean and the DREB gene derived from cotton. 本発明の実施例１の結果を示す図である。It is a figure which shows the result of Example 1 of this invention. 本発明の実施例１の結果を示す図である。It is a figure which shows the result of Example 1 of this invention. 本発明の実施例２の結果を示す図である。It is a figure which shows the result of Example 2 of this invention. 本発明の実施例２の結果を示す図である。It is a figure which shows the result of Example 2 of this invention.
発明の詳細な説明Detailed Description of the Invention
　現在、ＤＲＥＢ遺伝子等を活用した環境ストレスに強い作物の開発が世界的に行われており、今後ＤＲＥＢ遺伝子が導入された遺伝子組換え作物の生産が拡大することが予想される。したがって、ＤＲＥＢ遺伝子が導入された遺伝子組換え作物の検出技術が重要となる。しかし、ＤＲＥＢ遺伝子が導入された遺伝子組換え作物を、高い信頼性と実用性を持って検出可能な方法は未だ提供されていなかった。特許文献１にも、ＤＲＥＢ遺伝子を特異的に検出する方法については記載されていない。 Currently, the development of crops that are resistant to environmental stress using the DREB gene and the like is underway worldwide, and it is expected that the production of genetically modified crops into which the DREB gene has been introduced will be expanded in the future. Therefore, a technique for detecting a genetically modified crop into which the DREB gene has been introduced becomes important. However, there has not yet been provided a method that can detect a genetically modified crop into which the DREB gene has been introduced with high reliability and practicality. Patent Document 1 also does not describe a method for specifically detecting the DREB gene.
　したがって、本発明の目的は、ＤＲＥＢ遺伝子が導入された遺伝子組換え作物を、高い信頼性と実用性を持って検出可能な方法及び試薬キットを提供することにある。 Therefore, an object of the present invention is to provide a method and a reagent kit that can detect a genetically modified crop introduced with a DREB gene with high reliability and practicality.
　以下、本発明をその好ましい実施形態に基づいて説明する。
　本発明（第１及び第２発明）は、遺伝子組換え作物を検出する方法である。即ち、植物である作物、その作物から得られる食品素材、又は作物や食品素材を用いて得られる加工食品等の被検試料中に含まれる、組換えＤＮＡが導入された遺伝子組換え作物を検出する方法である。
　植物である作物としては、穀物粒、ジャガイモ、てんさい、パイナップル等が挙げられる。穀物粒としては、例えば、麦類（小麦、大麦、エンバク、ライ麦、ハトムギ等）、コメ、トウモロコシ、マイロ、アワ、ヒエ等のイネ科植物、ダイズ、小豆、落花生、エンドウマメ、インゲンマメ等のマメ科植物の種子が挙げられ、特に麦類であることが好ましい。その作物から得られる食品素材としては、作物が小麦である場合、薄力粉、中力粉、全粒粉、準強力粉、強力粉、デュラム小麦粉等が挙げられ、作物が小麦以外の穀物粒である場合、ライ麦粉、米粉等が挙げられる。作物やその作物から得られる食品素材を使用して得られる加工食品としては、作物が小麦である場合、パンや麺類、作物がダイズである場合、豆腐等が挙げられる。 Hereinafter, the present invention will be described based on preferred embodiments thereof.
The present invention (first and second inventions) is a method for detecting a genetically modified crop. That is, a genetically modified crop introduced with recombinant DNA contained in a test sample such as a plant crop, a food material obtained from the crop, or a processed food obtained using the crop or food material is detected. It is a method to do.
Examples of plant crops include cereal grains, potatoes, sugar beets, and pineapples. Grain grains include, for example, wheat (wheat, barley, oats, rye, barley, etc.), rice, corn, milo, millet, millet, and other legumes, soybeans, red beans, peanuts, peas, kidney beans, and other beans. The seed of a family plant is mentioned, It is especially preferable that it is wheat. Examples of food materials obtained from the crop include wheat flour, medium flour, whole grain flour, quasi-strong flour, strong flour, durum wheat flour, and rye flour when the crop is a grain other than wheat. , Rice flour and the like. Examples of processed foods obtained using crops and food materials obtained from the crops include tofu and the like when the crop is wheat, bread and noodles, and when the crop is soybean.
　本発明において検出する組換えＤＮＡは、ＤＲＥＢ遺伝子である。ＤＲＥＢ遺伝子は、作物の環境ストレス耐性を向上させ得るものであり、今後ＤＲＥＢ遺伝子が導入された遺伝子組換え作物の生産が拡大することが予想される。したがって、今後、遺伝子組換え作物の検出にＤＲＥＢ遺伝子を使用すれば、比較的簡便に遺伝子組換え作物を検出できるようになる可能性がある。なお、遺伝子組換え作物であるか否かの判定に、ＤＲＥＢ遺伝子の検査に加えて、ＤＲＥＢ遺伝子以外の組換えＤＮＡの検査を行うことも好ましい。 The recombinant DNA detected in the present invention is the DREB gene. The DREB gene can improve the environmental stress tolerance of crops, and it is expected that the production of genetically modified crops into which the DREB gene has been introduced will be expanded in the future. Therefore, if the DREB gene is used for detection of genetically modified crops in the future, it may be possible to detect genetically modified crops relatively easily. In addition to determining the DREB gene, it is also preferable to test a recombinant DNA other than the DREB gene in determining whether the plant is a genetically modified crop.
　第１発明においては、被検試料中のダイズ由来のＤＲＥＢ遺伝子をＰＣＲによって検出する。この検出は、被検試料中の核酸又は前記被検試料から抽出した核酸を鋳型とし、ダイズ由来のＤＲＥＢ遺伝子の部分配列を特異的に増幅可能なプライマーペアを用いて、ＰＣＲにより、ダイズ由来のＤＲＥＢ遺伝子の部分配列を有する核酸を増幅し、その増幅された核酸を検出又は定量する。
　第２発明においては、被検試料中のワタ由来のＤＲＥＢ遺伝子をＰＣＲによって検出する。この検出は、被検試料中の核酸又は前記被検試料から抽出した核酸を鋳型とし、ワタ由来のＤＲＥＢ遺伝子の部分配列を特異的に増幅可能なプライマーペアを用いて、ＰＣＲにより、ワタ由来のＤＲＥＢ遺伝子の部分配列を有する核酸を増幅し、その増幅された核酸を検出又は定量する。 In the first invention, the soybean-derived DREB gene in the test sample is detected by PCR. This detection is carried out by PCR using a primer pair that can specifically amplify a partial sequence of the DREB gene derived from soybean, using the nucleic acid in the sample or the nucleic acid extracted from the sample as a template. A nucleic acid having a partial sequence of the DREB gene is amplified, and the amplified nucleic acid is detected or quantified.
In the second invention, the cotton-derived DREB gene in the test sample is detected by PCR. This detection is performed by PCR using a primer pair that can specifically amplify a partial sequence of the DREB gene derived from cotton using a nucleic acid in the test sample or a nucleic acid extracted from the test sample as a template. A nucleic acid having a partial sequence of the DREB gene is amplified, and the amplified nucleic acid is detected or quantified.
　図１に、ダイズ由来のＤＲＥＢ遺伝子及びワタ由来のＤＲＥＢ遺伝子それぞれのＤＮＡ配列の一部を並べて示した。
　図１に示すように、ＤＲＥＢ遺伝子は、ダイズ由来のＤＲＥＢ遺伝子とワタ由来のＤＲＥＢ遺伝子のＤＮＡ配列が似ているため、第１発明においては、ダイズ由来のＤＲＥＢ遺伝子に特異的なＤＮＡ配列部分にプライマーを設計し、第２発明においては、ワタ由来のＤＲＥＢ遺伝子に特異的なＤＮＡ配列部分にプライマーを設計することが好ましい。 FIG. 1 shows part of the DNA sequence of each of the DREB gene derived from soybean and the DREB gene derived from cotton.
As shown in FIG. 1, in the DREB gene, the DNA sequence of the soybean-derived DREB gene is similar to that of the cotton-derived DREB gene. A primer is designed, and in the second invention, it is preferable to design a primer in a DNA sequence portion specific to the cotton-derived DREB gene.
　したがって、第１及び第３発明において、好ましいプライマーペアは、以下の（１）～（３）である。
（１）配列番号１に記載の塩基配列を含む核酸と、配列番号２に記載の塩基配列を含む核酸とからなるもの。配列番号１に記載の塩基配列は、配列番号１３に記載のダイズ由来のＤＲＥＢ遺伝子の全塩基配列中、図１にＲ１で示す領域の塩基配列であり、配列番号２に記載の塩基配列は、配列番号１３に記載のダイズ由来のＤＲＥＢ遺伝子の全塩基配列中、図１にＲ２で示す領域の相補鎖の塩基配列である。
（２）配列番号１に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸と、配列番号２に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸とからなるもの。
（３）配列番号１に記載の塩基配列と相補である配列番号３に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸と、配列番号２に記載の塩基配列と相補である配列番号４に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸とからなるもの。 Therefore, in the first and third inventions, preferred primer pairs are the following (1) to (3).
(1) A nucleic acid comprising the nucleic acid comprising the base sequence described in SEQ ID NO: 1 and the nucleic acid comprising the base sequence described in SEQ ID NO: 2. The base sequence described in SEQ ID NO: 1 is the base sequence of the region indicated by R1 in FIG. 1 in the entire base sequence of the soybean-derived DREB gene described in SEQ ID NO: 13, and the base sequence described in SEQ ID NO: 2 is It is the base sequence of the complementary strand of the region indicated by R2 in FIG. 1 in the entire base sequence of the soybean-derived DREB gene described in SEQ ID NO: 13.
(2) consisting of a nucleic acid containing at least 80% continuous base sequence in the base sequence described in SEQ ID NO: 1 and a nucleic acid containing at least 80% continuous base sequence in the base sequence shown in SEQ ID NO: 2 thing.
(3) a nucleic acid that hybridizes under stringent conditions to the base sequence described in SEQ ID NO: 3 that is complementary to the base sequence described in SEQ ID NO: 1, and a sequence number that is complementary to the base sequence described in SEQ ID NO: 2 A nucleic acid that hybridizes to the base sequence according to 4 under stringent conditions.
　また、ダイズ由来のＤＲＥＢ遺伝子とワタ由来のＤＲＥＢ遺伝子に共通なＤＮＡ配列部分にプライマーを設計することで、ダイズ由来及びワタ由来のＤＲＥＢ遺伝子の部分配列を有する核酸を同時に増幅することも可能である。本発明は、ダイズ由来のＤＲＥＢ遺伝子及びワタ由来のＤＲＥＢ遺伝子をそれぞれ特異的に、かつ同時に増幅させるプライマーペアを用いたＰＣＲによって、ダイズ由来又はワタ由来のＤＲＥＢ遺伝子が導入された遺伝子組換え作物を検出又は定量するものであってもよい。 It is also possible to simultaneously amplify a nucleic acid having a partial sequence of soybean-derived and cotton-derived DREB genes by designing a primer in a DNA sequence portion common to soybean-derived DREB gene and cotton-derived DREB gene. . The present invention relates to a genetically modified crop in which a soybean-derived or cotton-derived DREB gene is introduced by PCR using a primer pair that specifically and simultaneously amplifies soybean-derived DREB gene and cotton-derived DREB gene. You may detect or quantify.
　上記（１）のプライマーペアにおける、配列番号１に記載の塩基配列を含む核酸、上記（２）のプライマーペアにおける、配列番号１に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、及び上記（３）のプライマーペアにおける、配列番号１に記載の塩基配列と相補である配列番号３に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸は、それらのうちの何れか１以上と、上記（１）のプライマーペアにおける、配列番号２に記載の塩基配列を含む核酸、上記（２）のプライマーペアにおける、配列番号２に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、及び上記（３）のプライマーペアにおける、配列番号２に記載の塩基配列と相補である配列番号４に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸のうちの何れか１以上とを組み合わせて用いることもできる。本願の請求項２，８等の表現には、このような組み合わせからなるプライマーペアも含まれる。 A nucleic acid comprising the base sequence described in SEQ ID NO: 1 in the primer pair of (1) above, comprising at least 80% of the continuous base sequence in the base sequence described in SEQ ID NO: 1 in the primer pair of (2) above The nucleic acid and the nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 3 that is complementary to the nucleotide sequence of SEQ ID NO: 1 in the primer pair of (3) above are any of them. 1 or more and a nucleic acid comprising the base sequence described in SEQ ID NO: 2 in the primer pair of (1) above, and at least 80% continuous in the base sequence described in SEQ ID NO: 2 in the primer pair of (2) above The nucleic acid comprising the base sequence described above and the base sequence described in SEQ ID NO: 4 that is complementary to the base sequence described in SEQ ID NO: 2 in the primer pair of (3) above May be used in combination with stringent any one or more of the hybridizing nucleic acids under conditions. Expressions such as claims 2 and 8 of the present application include primer pairs composed of such combinations.
　同様に、第２及び第４発明において、好ましいプライマーペアは、以下の（４）～（６）である。
（４）配列番号６に記載の塩基配列を含む核酸と、配列番号７に記載の塩基配列を含む核酸とからなるもの。配列番号６に記載の塩基配列は、配列番号１４に記載のワタ由来のＤＲＥＢ遺伝子の全塩基配列中、図１にＲ６で示す領域の塩基配列であり、配列番号７に記載の塩基配列は、配列番号１４に記載のワタ由来のＤＲＥＢ遺伝子の塩基配列中、図１にＲ７で示す領域の相補鎖の塩基配列である。
（５）配列番号６に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸と、配列番号７に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸とからなるもの。
（６）配列番号６に記載の塩基配列と相補である配列番号８に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸と、配列番号７に記載の塩基配列と相補である配列番号９に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸とからなるもの。 Similarly, in the second and fourth inventions, preferred primer pairs are the following (4) to (6).
(4) A nucleic acid comprising the nucleic acid comprising the base sequence described in SEQ ID NO: 6 and the nucleic acid comprising the base sequence described in SEQ ID NO: 7. The base sequence described in SEQ ID NO: 6 is the base sequence of the region indicated by R6 in FIG. 1 in the entire base sequence of the cotton-derived DREB gene described in SEQ ID NO: 14, and the base sequence described in SEQ ID NO: 7 is It is the base sequence of the complementary strand of the region indicated by R7 in FIG. 1 in the base sequence of the cotton-derived DREB gene described in SEQ ID NO: 14.
(5) consisting of a nucleic acid comprising at least 80% continuous base sequence in the base sequence described in SEQ ID NO: 6 and a nucleic acid comprising at least 80% continuous base sequence in the base sequence described in SEQ ID NO: 7. thing.
(6) A nucleic acid that hybridizes under stringent conditions to the base sequence described in SEQ ID NO: 8 that is complementary to the base sequence described in SEQ ID NO: 6, and a sequence number that is complementary to the base sequence described in SEQ ID NO: 7 A nucleic acid that hybridizes to the base sequence according to 9 under stringent conditions.
　上記（４）のプライマーペアにおける、配列番号６に記載の塩基配列を含む核酸、上記（５）のプライマーペアにおける、配列番号６に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、及び上記（６）のプライマーペアにおける、配列番号６に記載の塩基配列と相補である配列番号８に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸は、それらのうちの何れか１以上と、上記（４）のプライマーペアにおける、配列番号７に記載の塩基配列を含む核酸、上記（５）のプライマーペアにおける、配列番号７に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、及び上記（６）のプライマーペアにおける、配列番号７に記載の塩基配列と相補である配列番号９に記載の塩基配列にストリンジェントな条件下でハイブリダイズする核酸のうちの何れか１以上とを組み合わせて用いることもできる。本願の請求項５，１０等の表現には、このような組み合わせからなるプライマーペアも含まれる。 A nucleic acid comprising the base sequence described in SEQ ID NO: 6 in the primer pair of (4) above, and at least 80% of the contiguous base sequence in the base sequence described in SEQ ID NO: 6 in the primer pair of (5) above The nucleic acid and the nucleic acid that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 8 that is complementary to the base sequence set forth in SEQ ID NO: 6 in the primer pair of (6) above are any of them. 1 or more and a nucleic acid comprising the base sequence described in SEQ ID NO: 7 in the primer pair of (4) above, and at least 80% continuous in the base sequence described in SEQ ID NO: 7 in the primer pair of (5) above The nucleic acid comprising the base sequence described above and the base sequence described in SEQ ID NO: 9 that is complementary to the base sequence described in SEQ ID NO: 7 in the primer pair of (6) above May be used in combination with stringent any one or more of the hybridizing nucleic acids under conditions. Expressions such as claims 5 and 10 of the present application include primer pairs composed of such combinations.
　上述した、配列番号１，３，６又は８に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む各核酸は、配列番号１，３，６又は８に記載の塩基配列中の８５％以上の連続した塩基配列を含む核酸であることが好ましく、配列番号１，３，６又は８に記載の塩基配列中の９０％以上の連続した塩基配列を含む核酸であることが更に好ましい。
　また、プライマーとして用いる核酸は、塩基数が１０以上であることが好ましく、１２以上であることがより好ましく、１５以上が更に好ましい。 Each nucleic acid containing at least 80% of the continuous base sequence in the base sequence described in SEQ ID NO: 1, 3, 6 or 8 is 85 in the base sequence described in SEQ ID NO: 1, 3, 6 or 8. It is preferable that the nucleic acid contains a continuous base sequence of at least 90%, more preferably a nucleic acid containing 90% or more of the continuous base sequence in the base sequence described in SEQ ID NO: 1, 3, 6 or 8.
The nucleic acid used as a primer preferably has 10 or more bases, more preferably 12 or more, and still more preferably 15 or more.
　上記（３）又は（６）のプライマーペアに関し、ストリンジェントな条件下とは、増幅対象のＤＮＡ配列と特異的なハイブリッドが形成され、かつ増幅対象でないＤＮＡ配列と非特異的なハイブリッドが形成されない条件をいう。また、（３）又は（６）のプライマーペアを構成する一対の核酸は、それぞれ相補の塩基配列にハイブリダイズした状態でプライマーとして機能する。ストリンジェントな条件は、例えば、ＤＮＡの長さに基づき、一般の技術を有する当業者によって、容易に決定することが可能である。当業者であれば、当該技術分野において公知の各種プライマー設計方法及びハイブリダイゼーション条件に関する技術常識、並びに通常用いられる実験手段を通じて得られる経験則を基に、選択されたプライマーに適切な条件を容易に見つけ出し、実施することができる。 With regard to the primer pair (3) or (6) above, under stringent conditions, a specific hybrid is formed with the DNA sequence to be amplified, and a non-specific hybrid is not formed with the DNA sequence that is not the amplification target. Say conditions. In addition, the pair of nucleic acids constituting the primer pair of (3) or (6) functions as a primer in a state where it is hybridized to a complementary base sequence. Stringent conditions can be readily determined by those skilled in the art based on the length of the DNA, for example. Those skilled in the art can easily determine the appropriate conditions for the selected primer based on the common knowledge of various primer design methods and hybridization conditions known in the art, and empirical rules obtained through commonly used experimental means. Can be found and implemented.
　このようなプライマーペアを用いてＰＣＲを行うことにより、第１発明においては、図１に示すダイズ由来のＤＲＥＢ遺伝子のＤＮＡ配列のうち、配列番号１１に記載の部分配列（図２参照）を特異的に増幅させることができ、第２発明においては、図１に示すワタ由来のＤＲＥＢ遺伝子のＤＮＡ配列のうち、配列番号１２（図３参照）に記載の部分配列を特異的に増幅させることができる。
　ここでいう特異的とは、第１及び第３発明においては、ダイズ由来のＤＲＥＢ遺伝子中の部分配列のみを増幅させることを意味し、第２及び第４発明においては、ワタ由来のＤＲＥＢ遺伝子中の部分配列のみを増幅させることを意味する。したがって、被験試料に含まれる、ダイズ由来のＤＲＥＢ遺伝子が導入されたダイズ以外の遺伝子組換え作物又はワタ由来のＤＲＥＢ遺伝子が導入されたワタ以外の遺伝子組換え作物をそれぞれ特異的に検出することができる。なお、第１及び第３発明において増幅させる部分配列は、配列番号１１であることが好ましいが、それに制限されるものではなく、ダイズ由来のＤＲＥＢ遺伝子のＤＮＡ配列の他の領域を増幅させてもよい。また、第２及び第４発明において増幅させる部分配列も、配列番号１２であることが好ましいが、それに制限されるものではなく、ワタ由来のＤＲＥＢ遺伝子のＤＮＡ配列の他の領域を増幅させてもよい。 By carrying out PCR using such a primer pair, in the first invention, the partial sequence shown in SEQ ID NO: 11 (see FIG. 2) of the DNA sequence of the soybean-derived DREB gene shown in FIG. In the second invention, the partial sequence described in SEQ ID NO: 12 (see FIG. 3) of the cotton-derived DREB gene DNA sequence shown in FIG. 1 can be specifically amplified. it can.
The term "specific" as used herein means that only the partial sequence in the soybean-derived DREB gene is amplified in the first and third inventions, and in the second and fourth inventions, in the cotton-derived DREB gene. Means that only a partial sequence of is amplified. Therefore, it is possible to specifically detect a genetically modified crop other than soybean introduced with a soybean-derived DREB gene or a genetically modified crop other than cotton introduced with a cotton-derived DREB gene, respectively, contained in a test sample. it can. Note that the partial sequence to be amplified in the first and third inventions is preferably SEQ ID NO: 11, but is not limited thereto, and other regions of the DNA sequence of the DREB gene derived from soybean may be amplified. Good. The partial sequence to be amplified in the second and fourth inventions is also preferably SEQ ID NO: 12, but is not limited thereto, and other regions of the cotton-derived DREB gene may be amplified. Good.
　本発明において、被検試料は、例えばそのまま又は粉砕して核酸抽出に供してもよく、洗浄し乾燥させた後破砕して核酸抽出に供してもよい。被検試料から抽出して分析に用いる核酸は、通常はＤＮＡである。ＤＮＡは公知の任意の方法によって抽出してもよいが、現在は多数のＤＮＡ抽出キットが市販されており、これらを用いて抽出することができる。例えばＤＮｅａｓｙ　Ｐｌａｎｔ　Ｍａｘｉ　ｋｉｔ（ＱＩＡＧＥＮ社製）を用いて、Kopellらの方法（Kopell, E. et al.; Mitteilungen aus dem Gebiete der Lebensmitteluntersuchung und Hygiene, 88, 164、出版社：Neukomm & Zimmermann）にしたがって、被検試料からＤＮＡを抽出してもよい。抽出したＤＮＡは、吸光度の測定などにより濃度を算出し、ＰＣＲに好適な濃度まで希釈して用いることが好ましい。 In the present invention, the test sample may be subjected to nucleic acid extraction, for example, as it is or after being pulverized, or may be washed, dried, crushed and subjected to nucleic acid extraction. The nucleic acid extracted from the test sample and used for analysis is usually DNA. DNA may be extracted by any known method, but many DNA extraction kits are commercially available and can be extracted using these. For example, using DNeasy Plant Maxi kit (manufactured by QIAGEN), using the method of Kopell et al. (Kopell, E. et al .; DNA may be extracted from the test sample. The extracted DNA is preferably used after calculating its concentration by measuring absorbance and diluting it to a concentration suitable for PCR.
　本発明において、ＰＣＲは、使用するプライマーやＤＮＡポリメラーゼを考慮して、常法にしたがって行うことができる。その際に、ＰＣＲ緩衝液、ｄＮＴＰ、及びＭｇＣｌ2等の試薬は調製してもよいし、市販のＰＣＲキットを用いてもよい。また、ＰＣＲ条件は、例えば９５℃１５秒、６０℃３０秒、及び７２℃３０秒を１サイクルとして３５サイクル行い、最後に終了反応として７２℃７分間という条件が使用可能であるが、用いるプライマーのＴｍ値、増幅すべき領域の長さ、及び鋳型ＤＮＡの濃度等を考慮して、適宜変更することができる。 In the present invention, PCR can be performed according to a conventional method in consideration of the primer and DNA polymerase to be used. At that time, reagents such as a PCR buffer, dNTP, and MgCl 2 may be prepared, or a commercially available PCR kit may be used. In addition, PCR conditions can be used, for example, at 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds for 35 cycles, and finally at 72 ° C. for 7 minutes as the end reaction. The Tm value, the length of the region to be amplified, the concentration of the template DNA, and the like can be changed as appropriate.
　増幅された核酸（以下「ＰＣＲ産物」という）は、特定のＤＮＡ断片を同定する任意の方法で検出できる。同定する方法としては、例えばアガロースゲル電気泳動、アクリルアミドゲル電気泳動、キャピラリー電気泳動、ハイブリダイゼーション、及び免疫学的方法等がある。一般的に、ＰＣＲ産物は電気泳動パターンによって同定される。例えばエチジウムブロミドを含む０．８％のアガロースゲルによる電気泳動を行い、ＰＣＲ産物をバンドとして検出してもよい。 The amplified nucleic acid (hereinafter referred to as “PCR product”) can be detected by any method for identifying a specific DNA fragment. Examples of the identification method include agarose gel electrophoresis, acrylamide gel electrophoresis, capillary electrophoresis, hybridization, and immunological methods. In general, PCR products are identified by electrophoresis patterns. For example, electrophoresis using 0.8% agarose gel containing ethidium bromide may be performed to detect the PCR product as a band.
　また、ＰＣＲ産物は、プライマーペア及び核酸プローブの存在下でリアルタイムＰＣＲを行い、ＰＣＲ増幅の過程で同定することもできる。また、リアルタイムＰＣＲで使用する核酸プローブは蛍光標識、放射性物質標識、及びビオチン標識されたものを用いることができる。本発明で使用する核酸プローブは、例えば、配列番号５又は配列番号１０に記載の塩基配列からなるオリゴヌクレオチドの５’末端を蛍光物質としてＦＡＭで修飾し、３’末端をクエンチャーとしてＴＡＭＲＡ又はＭＧＢで修飾したものである。また、配列番号５又は配列番号１０に記載の塩基配列に代えて、配列番号５又は配列番号１０に記載の塩基配列と相補の塩基配列からなる核酸プローブや、配列番号５若しくは配列番号１０又はこれらと相補の塩基配列に対して８０％以上、より好ましくは８５％以上、更に好ましくは９０％以上の相同性を有する塩基配列を含み、蛍光標識、放射性物質標識又はビオチン標識されている核酸プローブを用いることもできる。 The PCR product can also be identified in the process of PCR amplification by performing real-time PCR in the presence of a primer pair and a nucleic acid probe. In addition, as a nucleic acid probe used in real-time PCR, fluorescent label, radioactive substance label, and biotin-labeled probe can be used. The nucleic acid probe used in the present invention is, for example, modified with FAM using the 5 ′ end of an oligonucleotide having the base sequence described in SEQ ID NO: 5 or SEQ ID NO: 10 as a fluorescent substance, and TAMRA or MGB using the 3 ′ end as a quencher. It is modified with Further, in place of the base sequence described in SEQ ID NO: 5 or SEQ ID NO: 10, a nucleic acid probe consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 5 or SEQ ID NO: 10, SEQ ID NO: 5 or SEQ ID NO: 10 or these A nucleic acid probe comprising a nucleotide sequence having a homology of 80% or more, more preferably 85% or more, more preferably 90% or more, and a fluorescent label, radioactive substance label or biotin label It can also be used.
　第３発明及び第４発明は、それぞれ第１発明及び第２発明で使用したプライマーペアを含む試薬キットである。プライマーペアを構成する核酸は常法にしたがって製造することができる。また、試薬キットはプライマーペアと他の試薬とを含んでいてもよい。例えば、試薬キットはｄＮＴＰ、ＭｇＣｌ2、ＤＮＡポリメラーゼ（例えばＴａｑＤＮＡポリメラーゼ）、緩衝液（例えばＴｒｉｓ－ＨＣｌ）、グリセロール、ＤＭＳＯ、ポジティブコントロール用ＤＮＡ、ネガティブコントロール用ＤＮＡ、及び蒸留水等を包含してもよい。試薬キットは、ＰＣＲによる増幅産物の核酸の検出又は定量に使用するための、蛍光標識、放射性物質標識又はビオチン標識されている核酸プローブを含んでいてもよい。試薬キットに含まれる試薬は、それぞれ独立に梱包されていてもよいし、混合された上で梱包されていてもよい。試薬キット中のそれぞれの試薬濃度に特に制限はなく、本発明に係るＰＣＲが実施可能な濃度範囲であればよい。また、試薬キットには、好適なＰＣＲ条件等の情報がさらに添付されていてもよい。 The third invention and the fourth invention are reagent kits including the primer pairs used in the first invention and the second invention, respectively. The nucleic acid constituting the primer pair can be produced according to a conventional method. The reagent kit may contain a primer pair and another reagent. For example, the reagent kit may include dNTP, MgCl 2 , DNA polymerase (eg, Taq DNA polymerase), buffer (eg, Tris-HCl), glycerol, DMSO, positive control DNA, negative control DNA, distilled water, and the like. Good. The reagent kit may include a fluorescent probe, a radioactive substance label, or a biotin-labeled nucleic acid probe for use in the detection or quantification of the nucleic acid of the amplification product by PCR. Reagents contained in the reagent kit may be individually packed, or may be packed after being mixed. There are no particular limitations on the concentration of each reagent in the reagent kit, and any concentration range can be used as long as PCR according to the present invention can be performed. Further, information such as suitable PCR conditions may be further attached to the reagent kit.
　以下、実施例により本発明を更に詳細に説明する。本発明は、以下の実施例により何ら制限されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited in any way by the following examples.
〔実施例１：ＰＣＲ及びアガロースゲル電気泳動によるダイズ由来及びワタ由来のＤＲＥＢ遺伝子の検出〕
（方法）
　下記の３種の被検試料からそれぞれ抽出したＤＮＡを鋳型とし、下記のプライマーペアを用いてＰＣＲを行った。その後、アガロースゲル電気泳動により各試料における増幅の有無を調べた。
（試料）
　小麦：国内産小麦
　ダイズ：市販の国内産ダイズの乾燥種子
　ワタ：市販のワタの乾燥種子
　各試料をＭｕｌｔｉ　Ｂｅａｄｓ　Ｓｈｏｃｋｅｒ（安井器械株式会社）で破砕した後、ＤＮｅａｓｙ　Ｐｌａｎｔ　Ｍａｘｉ　ｋｉｔ(ＱＩＡＧＥＮ社製)を用いてＤＮＡを抽出した。 [Example 1: Detection of soybean-derived and cotton-derived DREB genes by PCR and agarose gel electrophoresis]
(Method)
PCR was carried out using the DNA extracted from each of the following three test samples as a template and using the following primer pairs. Thereafter, the presence or absence of amplification in each sample was examined by agarose gel electrophoresis.
(sample)
Wheat: Domestic wheat Soybean: Dry domestic seeds of commercially available soybeans Cotton: Dry seeds of commercially available cotton After each sample was crushed with Multi Beads Shocker (Yasui Kikai Co., Ltd.), DNeasy Plant Maxi kit (QIAGEN) was used. DNA was extracted.
（プライマーペア）
　ＰＣＲに使用するプライマーを設計するために、遺伝子配列データベース（Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ）（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／）を使用した。
　ダイズ由来のＤＲＥＢ遺伝子のＤＮＡ配列としてａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　ＡＦ５１４９０８（配列番号１３）を使用し、配列番号１に記載の塩基配列を有する核酸と配列番号２に記載の塩基配列を有する核酸とからなるプライマーペアを設計した。
　ワタ由来のＤＲＥＢ遺伝子のＤＮＡ配列としてａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ　ＡＦ５０９５０２（配列番号１４）を使用し、配列番号６に記載の塩基配列を有する核酸と配列番号７に記載の塩基配列を有する核酸とからなるプライマーペアを設計した。
　プライマーの合成は、Ｏｐｅｒｏｎ社に委託した。 (Primer pair)
In order to design primers used for PCR, a gene sequence database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/) was used.
Using accession number AF514908 (SEQ ID NO: 13) as the DNA sequence of the DREB gene derived from soybean, a primer pair consisting of a nucleic acid having the base sequence described in SEQ ID NO: 1 and a nucleic acid having the base sequence described in SEQ ID NO: 2 Designed.
Using accession number AF509502 (SEQ ID NO: 14) as the DNA sequence of the DREB gene derived from cotton, a primer pair comprising a nucleic acid having the base sequence described in SEQ ID NO: 6 and a nucleic acid having the base sequence described in SEQ ID NO: 7 Designed.
Primer synthesis was outsourced to Operon.
（ＰＣＲによるダイズ由来及びワタ由来のＤＲＥＢ遺伝子の検出）
　各試料から抽出したＤＮＡを鋳型にＰＣＲを行った。ダイズ由来のＤＲＥＢ遺伝子の検出には配列番号１及び配列番号２のプライマーを使用した。また、ワタ由来のＤＲＥＢ遺伝子の検出には配列番号６及び配列番号７のプライマーを使用した。
　３種の被験試料のそれぞれについて、ＰＣＲの反応ミックスを調整した。１μｌのＤＮＡ（２０　ｎｇ／μｌ）、０．５μｌ（２．５Ｕ）のＡｍｐｌｉＴａｑ　Ｇｏｌｄ(Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ)、２．５μｌの２　ｍＭ　ｄＮＴＰ、２．５μｌの２５　ｍＭ　ＭｇＣｌ2、０．１５μｌの５０μＭ各プライマー、２．５μｌの１０×ＰＣＲ　Ｂｕｆｆｅｒ　ＩＩを添加し、さらに、反応ミックスの液量が２５μｌとなるよう、蒸留水を添加した。
　ＰＣＲにおいては、９５℃１０分のＤＮＡポリメラーゼ活性化ステップを実施した後、９５℃１５秒の変性ステップ、６０℃３０秒のアニーリングステップ及び７２℃３０秒のＤＮＡ伸長ステップを含むサイクルを３５回繰り返し、その後、７２℃５分のＤＮＡ伸長最終ステップを行った。 (Detection of DREB genes derived from soybean and cotton by PCR)
PCR was performed using DNA extracted from each sample as a template. Primers of SEQ ID NO: 1 and SEQ ID NO: 2 were used for detection of the DREB gene derived from soybean. In addition, primers of SEQ ID NO: 6 and SEQ ID NO: 7 were used for detection of the cotton-derived DREB gene.
The PCR reaction mix was adjusted for each of the three test samples. 1 μl DNA (20 ng / μl), 0.5 μl (2.5 U) AmpliTaq Gold (Applied Biosystems), 2.5 μl 2 mM dNTP, 2.5 μl 25 mM MgCl 2 , 0.15 μl 50 μM each primer Then, 2.5 μl of 10 × PCR Buffer II was added, and distilled water was further added so that the volume of the reaction mix was 25 μl.
In PCR, after performing a DNA polymerase activation step at 95 ° C. for 10 minutes, a cycle including a denaturation step at 95 ° C. for 15 seconds, an annealing step at 60 ° C. for 30 seconds and a DNA extension step at 72 ° C. for 30 seconds is repeated 35 times. Thereafter, a final DNA extension step at 72 ° C. for 5 minutes was performed.
　ＰＣＲ終了後、ＰＣＲ反応溶液５μｌをＥ－Ｇｅｌ　ＥＸ（インビトロジェン）にて電気泳動を行い、ＵＶで増幅を確認した。
　増幅が確認されたＰＣＲ産物を精製した後、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒｓ　ｖ１．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ（ライフテクノロジーズジャパン）を使用してサイクルシーケンス反応を行った。シーケンス反応物を精製した後、Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ３５００ジェネティックアナライザ（ライフテクノロジーズジャパン）にてＤＮＡ配列を調べた。 After completion of PCR, 5 μl of the PCR reaction solution was electrophoresed with E-Gel EX (Invitrogen), and amplification was confirmed by UV.
After the PCR product confirmed to be amplified was purified, a cycle sequence reaction was performed using BigDye Terminators v1.1 Cycle Sequencing Kit (Life Technologies Japan). After purifying the sequencing reaction product, the DNA sequence was examined with Applied Biosystems 3500 Genetic Analyzer (Life Technologies Japan).
（結果）
　ダイズ由来のＤＲＥＢ遺伝子の検出結果を図２に示した。図２に示すように、ダイズから抽出したＤＮＡのみ予想された長さ（９１ｂｐ）のバンドが検出され、ワタ及び小麦から抽出したＤＮＡではバンドは検出されなかった。さらに、ダイズで検出されたバンドのＤＮＡ配列は、ダイズ由来のＤＲＥＢ遺伝子の増幅領域の配列であった。
　ワタ由来のＤＲＥＢ遺伝子の検出結果を図３に示した。図３に示すように、ワタから抽出したＤＮＡのみ予想された長さ（９４ｂｐ）のバンドが検出され、ダイズ及び小麦から抽出したＤＮＡではバンドは検出されなかった。さらに、ワタで検出されたバンドのＤＮＡ配列は、ワタ由来のＤＲＥＢ遺伝子の増幅領域の配列であった。なお、図２及び図３の「４．ＴＥ」はネガティブコントロールである。 (result)
The detection result of the soybean-derived DREB gene is shown in FIG. As shown in FIG. 2, a band of the expected length (91 bp) was detected only for DNA extracted from soybean, and no band was detected for DNA extracted from cotton and wheat. Furthermore, the DNA sequence of the band detected in soybean was the sequence of the amplified region of the DREB gene derived from soybean.
The detection result of cotton-derived DREB gene is shown in FIG. As shown in FIG. 3, a band of the expected length (94 bp) was detected only for DNA extracted from cotton, and no band was detected for DNA extracted from soybean and wheat. Furthermore, the DNA sequence of the band detected in cotton was the sequence of the amplified region of the cotton-derived DREB gene. Note that “4.TE” in FIGS. 2 and 3 is a negative control.
　これらの結果より、配列番号１、２及び配列番号６、７のプライマーペアを用いてＰＣＲを行うことで、ダイズ由来及びワタ由来のＤＲＥＢ遺伝子をそれぞれ特異的に増幅できることがわかった。したがって、これらのプライマーペアを使用すれば、それぞれダイズ由来及びワタ由来のＤＲＥＢ遺伝子が導入された、ダイズ以外及びワタ以外の遺伝子組換え作物を検出することができる。また、これらのプライマーペアは小麦では増幅しないことから、ダイズ由来及びワタ由来のＤＲＥＢ遺伝子が導入された遺伝子組換え小麦を検出することができる。 From these results, it was found that the DREB genes derived from soybean and cotton can be specifically amplified by PCR using the primer pairs of SEQ ID NOs: 1, 2 and SEQ ID NOs: 6, 7. Therefore, if these primer pairs are used, genetically modified crops other than soybean and cotton other than soybean introduced with soybean-derived and cotton-derived DREB genes can be detected. Moreover, since these primer pairs are not amplified in wheat, it is possible to detect genetically modified wheat introduced with soybean-derived and cotton-derived DREB genes.
〔実施例２：リアルタイムＰＣＲによるダイズ由来及びワタ由来のＤＲＥＢ遺伝子の検出〕
（方法）
　実施例１で使用したものと同じ３種の被検試料からそれぞれ抽出したＤＮＡを鋳型とし、下記のプライマーペア及び核酸プローブを用いてリアルタイムＰＣＲを行った。
（プライマーペア）
　ダイズ由来のＤＲＥＢ遺伝子においては、実施例１で使用した配列番号１、２に記載の塩基配列を有する核酸からなるプライマーペアを用いた。
　ワタ由来のＤＲＥＢ遺伝子においては、実施例１で使用した配列番号６、７に記載の塩基配列を有する核酸からなるプライマーペアを用いた。
（核酸プローブ）
　ダイズ由来のＤＲＥＢ遺伝子においては、プライマーペア（配列番号１、２）の間のＤＮＡ配列に配列番号５の核酸プローブを設計した。この核酸プローブは５’末端をＦＡＭで標識、３’末端をクエンチャーとしてＴＡＭＲＡで修飾した。
　ワタ由来のＤＲＥＢ遺伝子においては、プライマーペア（配列番号６、７）の間のＤＮＡ配列に配列番号１０の核酸プローブを設計した。この核酸プローブは５’末端をＦＡＭで標識、３’末端をクエンチャーとしてＭＧＢで修飾した。
　核酸プローブの合成は、Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社に委託した。 [Example 2: Detection of soybean-derived and cotton-derived DREB genes by real-time PCR]
(Method)
Real-time PCR was performed using DNA extracted from the same three types of test samples as used in Example 1 as templates and using the following primer pairs and nucleic acid probes.
(Primer pair)
For the DREB gene derived from soybean, a primer pair consisting of a nucleic acid having the base sequence described in SEQ ID NOs: 1 and 2 used in Example 1 was used.
In the cotton-derived DREB gene, a primer pair consisting of nucleic acids having the base sequences described in SEQ ID NOs: 6 and 7 used in Example 1 was used.
(Nucleic acid probe)
For the soybean-derived DREB gene, the nucleic acid probe of SEQ ID NO: 5 was designed in the DNA sequence between the primer pair (SEQ ID NOs: 1, 2). This nucleic acid probe was labeled with FAM at the 5 ′ end and modified with TAMRA using the 3 ′ end as a quencher.
For the cotton-derived DREB gene, a nucleic acid probe of SEQ ID NO: 10 was designed in the DNA sequence between the primer pair (SEQ ID NOs: 6, 7). This nucleic acid probe was labeled with FAM at the 5 ′ end and modified with MGB using the 3 ′ end as a quencher.
The synthesis of the nucleic acid probe was outsourced to Applied Biosystems.
（リアルタイムＰＣＲによるダイズ由来及びワタ由来のＤＲＥＢ遺伝子の検出）
　各試料から抽出したＤＮＡを鋳型にリアルタイムＰＣＲを行った。ダイズ由来のＤＲＥＢ遺伝子の検出には配列番号１及び配列番号２のプライマー、配列番号５のオリゴヌクレオチドを含む蛍光標識された核酸プローブを使用した。また、ワタ由来のＤＲＥＢ遺伝子の検出には配列番号６及び配列番号７のプライマー、配列番号１０のオリゴヌクレオチドを含む蛍光標識された核酸プローブを使用した。３種の被験試料のそれぞれについて、リアルタイムＰＣＲの反応ミックスを調整した。２．５μｌのＤＮＡ（２０　ｎｇ／μｌ）、１２．５μｌのＴａｑＭａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）、０．２５μｌの５０μＭ各プライマー、０．５μｌの１０μＭ核酸プローブを添加し、さらに、反応ミックスの液量が２５μｌとなるよう、蒸留水を添加した。
　リアルタイムＰＣＲにおいては、７９００ＨＴ　Ｆａｓｔ　Ｒｅａｌ－Ｔｉｍｅ　ＰＣＲ　Ｓｙｓｔｅｍ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）を使用した。５０℃２分に続いて９５℃１０分の変性ステップを実施した後、９５℃１５秒の変性ステップ及び６０℃１分のアニーリング及びＤＮＡ伸長ステップを含むサイクルを４５サイクル繰り返した。
　リアルタイムＰＣＲ終了後、各試料において、ＦＡＭ由来の蛍光強度が指数関数的に増加しているか確認した。 (Detection of soybean and cotton-derived DREB genes by real-time PCR)
Real-time PCR was performed using DNA extracted from each sample as a template. For detection of soybean-derived DREB gene, a fluorescently labeled nucleic acid probe containing the primers of SEQ ID NO: 1 and SEQ ID NO: 2 and the oligonucleotide of SEQ ID NO: 5 was used. For detection of the cotton-derived DREB gene, a fluorescently labeled nucleic acid probe containing the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and the oligonucleotide of SEQ ID NO: 10 was used. A real-time PCR reaction mix was prepared for each of the three test samples. 2.5 μl of DNA (20 ng / μl), 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems), 0.25 μl of 50 μM each primer, 0.5 μl of 10 μM nucleic acid probe were added, and the reaction mix Distilled water was added so that the liquid volume was 25 μl.
For real-time PCR, 7900HT Fast Real-Time PCR System (Applied Biosystems) was used. After performing a denaturation step at 95 ° C. for 10 minutes following 50 ° C. for 2 minutes, a cycle including a denaturation step at 95 ° C. for 15 seconds and an annealing and DNA extension step at 60 ° C. for 1 minute was repeated 45 cycles.
After completion of real-time PCR, it was confirmed in each sample whether the fluorescence intensity derived from FAM increased exponentially.
（結果）
　ダイズ由来のＤＲＥＢ遺伝子の検出結果を図４に示した。図４に示すように、ダイズから抽出したＤＮＡのみ指数関数的に増加した蛍光強度が検出され、ワタ及び小麦から抽出したＤＮＡでは検出されなかった。
　ワタ由来のＤＲＥＢ遺伝子の検出結果を図５に示した。図５に示すように、ワタから抽出したＤＮＡのみ指数関数的に増加した蛍光強度が検出され、ダイズ及び小麦から抽出したＤＮＡでは検出されなかった。 (result)
The detection result of the soybean-derived DREB gene is shown in FIG. As shown in FIG. 4, the fluorescence intensity increased exponentially only with DNA extracted from soybean, but not with DNA extracted from cotton and wheat.
The detection result of cotton-derived DREB gene is shown in FIG. As shown in FIG. 5, the fluorescence intensity increased exponentially only with DNA extracted from cotton, but not with DNA extracted from soybean and wheat.
　これらの結果より、配列番号１、２のプライマーペアと配列番号５のオリゴヌクレオチドを含む蛍光標識された核酸プローブ、及び配列番号６、７のプライマーペアと配列番号１０のオリゴヌクレオチドを含む蛍光標識された核酸プローブを用いてリアルタイムＰＣＲを行うことで、ダイズ由来及びワタ由来のＤＲＥＢ遺伝子をそれぞれ特異的に検出できることがわかった。したがって、これらのプライマーペア及び核酸プローブのセットを使用すれば、リアルタイムＰＣＲにおいてもそれぞれダイズ由来及びワタ由来のＤＲＥＢ遺伝子が導入された、ダイズ以外及びワタ以外の遺伝子組換え作物を検出することができる。また、これらのプライマーペア及び核酸プローブのセットは小麦では増幅しないことから、ダイズ由来及びワタ由来のＤＲＥＢ遺伝子が導入された遺伝子組換え小麦を検出することができる。 From these results, the fluorescently labeled nucleic acid probe containing the primer pair of SEQ ID NO: 1 and 2 and the oligonucleotide of SEQ ID NO: 5 and the fluorescently labeled nucleic acid probe containing the primer pair of SEQ ID NO: 6, 7 and the oligonucleotide of SEQ ID NO: 10 were labeled. It was found that the DREB gene derived from soybean and cotton can be specifically detected by performing real-time PCR using the nucleic acid probe. Therefore, if these primer pairs and nucleic acid probe sets are used, it is possible to detect genetically modified crops other than soybean and cotton other than soybean and cotton-derived DREB genes introduced in real-time PCR, respectively. . Moreover, since these primer pairs and nucleic acid probe sets are not amplified in wheat, it is possible to detect genetically modified wheat introduced with soybean-derived and cotton-derived DREB genes.
　本発明の遺伝子組換え作物の検出方法及び試薬キットによれば、農産物やそれを使用した食品等の被検試料中に含まれる、ＤＲＥＢ遺伝子が導入された遺伝子組換え作物を、高い信頼性及び実用性を持って検出可能である。 According to the method and reagent kit for detecting a genetically modified crop of the present invention, a genetically modified crop introduced with a DREB gene, which is contained in a test sample such as an agricultural product or a food using the same, has high reliability and It can be detected with practicality.

Claims (12)

  1. 　遺伝子組換え作物をポリメラーゼ連鎖反応によって検出する遺伝子組換え作物の検出方法であって、
    　被検試料中の核酸又は前記被検試料から抽出した核酸を鋳型とし、ダイズ由来のＤＲＥＢ遺伝子の部分配列を特異的に増幅可能なプライマーペアを用いて、前記ダイズ由来のＤＲＥＢ遺伝子の部分配列を有する核酸を増幅する工程と、
    　前記増幅された核酸を検出又は定量する工程と、
    　を含む遺伝子組換え作物の検出方法。     A method for detecting a genetically modified crop by detecting a genetically modified crop by polymerase chain reaction,
    Using a nucleic acid in a test sample or a nucleic acid extracted from the test sample as a template, and using a primer pair that can specifically amplify a partial sequence of the soybean-derived DREB gene, the partial sequence of the soybean-derived DREB gene Amplifying the nucleic acid possessed;
    Detecting or quantifying the amplified nucleic acid;
    A method for detecting a genetically modified crop comprising
  2. 　前記プライマーペアが、
    　配列番号１に記載の塩基配列を含む核酸、
    　配列番号１に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は　配列番号３に記載の塩基配列を含む核酸にハイブリダイズする核酸と、
    　配列番号２に記載の塩基配列を含む核酸、
    　配列番号２に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は　配列番号４に記載の塩基配列を含む核酸にハイブリダイズする核酸とからなる、請求項１に記載の遺伝子組換え作物の検出方法。     The primer pair is
    A nucleic acid comprising the base sequence set forth in SEQ ID NO: 1,
    A nucleic acid containing at least 80% of the continuous base sequence in the base sequence shown in SEQ ID NO: 1, or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 3,
    A nucleic acid comprising the base sequence set forth in SEQ ID NO: 2,
    The gene according to claim 1, comprising a nucleic acid containing at least 80% of the continuous base sequence of the base sequence shown in SEQ ID NO: 2 or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 4. Method for detecting recombinant crops.
  3. 　前記増幅された核酸の検出又は定量に、配列番号５に記載の塩基配列、該塩基配列と相補の塩基配列又はこれらの何れかの塩基配列に対して８０％以上の相同性を有する塩基配列を含み、蛍光標識、放射性物質標識又はビオチン標識されている核酸プローブを用いる、請求項１又は２に記載の遺伝子組換え作物の検出方法。 For detection or quantification of the amplified nucleic acid, the base sequence set forth in SEQ ID NO: 5, the base sequence complementary to the base sequence, or a base sequence having 80% or more homology with any of these base sequences The method for detecting a genetically modified crop according to claim 1 or 2, wherein a nucleic acid probe containing a fluorescent label, a radioactive substance label or a biotin label is used.
  4. 　遺伝子組換え作物をポリメラーゼ連鎖反応によって検出する遺伝子組換え作物の検出方法であって、
    　被検試料中の核酸又は前記被検試料から抽出した核酸を鋳型とし、ワタ由来のＤＲＥＢ遺伝子の部分配列を特異的に増幅可能なプライマーペアを用いて、前記ワタ由来のＤＲＥＢ遺伝子の部分配列を有する核酸を増幅する工程と、
    　前記増幅された核酸を検出又は定量する工程と、
    　を含む遺伝子組換え作物の検出方法。     A method for detecting a genetically modified crop by detecting a genetically modified crop by polymerase chain reaction,
    Using a nucleic acid in a test sample or a nucleic acid extracted from the test sample as a template, and using a primer pair that can specifically amplify a partial sequence of a cotton-derived DREB gene, a partial sequence of the cotton-derived DREB gene Amplifying the nucleic acid possessed;
    Detecting or quantifying the amplified nucleic acid;
    A method for detecting a genetically modified crop comprising
  5. 　前記プライマーペアが、
    　配列番号６に記載の塩基配列を含む核酸、
    　配列番号６に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は　配列番号８に記載の塩基配列を含む核酸にハイブリダイズする核酸と、
    　配列番号７に記載の塩基配列を含む核酸、
    　配列番号７に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は　配列番号９に記載の塩基配列を含む核酸にハイブリダイズする核酸とからなる、請求項４に記載の遺伝子組換え作物の検出方法。     The primer pair is
    A nucleic acid comprising the base sequence set forth in SEQ ID NO: 6,
    A nucleic acid containing at least 80% of the continuous base sequence in the base sequence shown in SEQ ID NO: 6 or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 8,
    A nucleic acid comprising the base sequence set forth in SEQ ID NO: 7,
    The gene according to claim 4, comprising a nucleic acid containing at least 80% of the continuous base sequence of the base sequence shown in SEQ ID NO: 7 or a nucleic acid hybridizing to a nucleic acid containing the base sequence shown in SEQ ID NO: 9. Method for detecting recombinant crops.
  6. 　前記増幅された核酸の検出又は定量に、配列番号１０に記載の塩基配列、該塩基配列と相補の塩基配列又はこれらの何れかの塩基配列に対して８０％以上の相同性を有する塩基配列を含み、蛍光標識、放射性物質標識又はビオチン標識されている核酸プローブを用いる、請求項４又は５に記載の遺伝子組換え作物の検出方法。 For detection or quantification of the amplified nucleic acid, a base sequence represented by SEQ ID NO: 10, a base sequence complementary to the base sequence, or a base sequence having 80% or more homology with any of these base sequences The method for detecting a genetically modified crop according to claim 4 or 5, wherein a nucleic acid probe containing a fluorescent label, a radioactive substance label or a biotin label is used.
  7. 　前記遺伝子組換え作物が小麦である、請求項１～６の何れか１項に記載の遺伝子組換え作物の検出方法。 The method for detecting a genetically modified crop according to any one of claims 1 to 6, wherein the genetically modified crop is wheat.
  8. 　ダイズ由来のＤＲＥＢ遺伝子が導入された遺伝子組換え作物を、ポリメラーゼ連鎖反応によって検出又は定量するための試薬キットであって、
    　配列番号１に記載の塩基配列を含む核酸、配列番号１に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号３に記載の塩基配列を含む核酸にハイブリダイズする核酸と、
    　配列番号２に記載の塩基配列を含む核酸、配列番号２に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号４に記載の塩基配列を含む核酸にハイブリダイズする核酸とからなるプライマーペアを含む、試薬キット。     A reagent kit for detecting or quantifying a genetically modified crop introduced with soybean-derived DREB gene by polymerase chain reaction,
    Hybridizes to a nucleic acid comprising the base sequence set forth in SEQ ID NO: 1, a nucleic acid comprising at least 80% of the contiguous base sequence in the base sequence set forth in SEQ ID NO: 1, or a nucleic acid comprising the base sequence set forth in SEQ ID NO: 3. Nucleic acids,
    It hybridizes to a nucleic acid comprising the base sequence set forth in SEQ ID NO: 2, a nucleic acid comprising at least 80% continuous base sequence in the base sequence set forth in SEQ ID NO: 2, or a nucleic acid comprising the base sequence set forth in SEQ ID NO: 4. A reagent kit comprising a primer pair consisting of a nucleic acid.
  9. 　配列番号５に記載の塩基配列、該塩基配列と相補の塩基配列又はこれらの何れかの塩基配列に対して８０％以上の相同性を有する塩基配列を含み、蛍光標識、放射性物質標識又はビオチン標識されている核酸プローブを更に含む、請求項８に記載の試薬キット。 Including a nucleotide sequence set forth in SEQ ID NO: 5, a nucleotide sequence complementary to the nucleotide sequence, or a nucleotide sequence having 80% or more homology with any of these nucleotide sequences, and a fluorescent label, radioactive substance label, or biotin label The reagent kit according to claim 8, further comprising a nucleic acid probe.
  10. 　ワタ由来のＤＲＥＢ遺伝子が導入された遺伝子組換え作物を、ポリメラーゼ連鎖反応によって検出又は定量するための試薬キットであって、
    　配列番号６に記載の塩基配列を含む核酸、配列番号６に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号８に記載の塩基配列を含む核酸にハイブリダイズする核酸と、
    　配列番号７に記載の塩基配列を含む核酸、配列番号７に記載の塩基配列中の少なくとも８０％の連続した塩基配列を含む核酸、又は配列番号９に記載の塩基配列を含む核酸にハイブリダイズする核酸とからなるプライマーペアを含む、試薬キット。     A reagent kit for detecting or quantifying a genetically modified crop introduced with cotton-derived DREB gene by polymerase chain reaction,
    It hybridizes to a nucleic acid comprising the base sequence set forth in SEQ ID NO: 6, a nucleic acid comprising at least 80% of the contiguous base sequence in the base sequence set forth in SEQ ID NO: 6, or a nucleic acid comprising the base sequence set forth in SEQ ID NO: 8. Nucleic acids,
    It hybridizes to a nucleic acid comprising the base sequence set forth in SEQ ID NO: 7, a nucleic acid comprising at least 80% continuous base sequence in the base sequence set forth in SEQ ID NO: 7, or a nucleic acid comprising the base sequence set forth in SEQ ID NO: 9. A reagent kit comprising a primer pair consisting of a nucleic acid.
  11. 　配列番号１０に記載の塩基配列、該塩基配列と相補の塩基配列又はこれらの何れかの塩基配列に対して８０％以上の相同性を有する塩基配列を含み、蛍光標識、放射性物質標識又はビオチン標識されている核酸プローブを更に含む、請求項１０に記載の試薬キット。 Including a nucleotide sequence set forth in SEQ ID NO: 10, a nucleotide sequence complementary to the nucleotide sequence, or a nucleotide sequence having 80% or more homology with any of these nucleotide sequences, and a fluorescent label, radioactive substance label, or biotin label The reagent kit according to claim 10, further comprising a nucleic acid probe.
  12. 　前記遺伝子組換え作物が小麦である、請求項８～１１の何れか１項に記載の試薬キット。 The reagent kit according to any one of claims 8 to 11, wherein the genetically modified crop is wheat.
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