KR100773085B1 - Method for analyzing genes from transgenic plants - Google Patents

Abstract

The present invention relates to an analytical method for analyzing a stilbene synthetic gene from a transformed sulfur ( Rhemannia glutinosa L.) plant to produce a stilbene compound, more specifically, the stellbene synthesis symbolizing the synthesis of resveratrol and related derivatives The present invention relates to a gene analysis method using a DNA chip capable of confirming expression of a stilbene gene from a mutant transformed plant of a gene and confirming a change in expression patterns of other genes in the transformed plant. Gene analysis method using the DNA chip of the present invention can quickly test whether the gene is expressed by using the active site of any gene in the organism as a probe (probe), the gene is introduced Since a transgenic plant can be used to compare expression patterns of a plurality of genes, it can be usefully used for analyzing the expression patterns of the genes and other genes in a plant into which the modified gene is inserted.

DNA chip, probe DNA, gene analysis.

Description

Method for analyzing genes from transgenic plants             

1 is a conceptual diagram showing in detail a process of analyzing a gene encoding a stilbene synthase from a sulfur-like plant which has transduced a gene encoding a stilbene syngene using a DNA chip.

2 is a method for retrieving amino acid sequences for genes capable of synthesizing resveratrol-type compounds using a PROSITE program (http://us.expasy.org/) and determining sequences Method and amplification of stilbene synthase and key enzymes of other genes using PCR, and amplifying the amplified PCR products on a substrate (glass), hybridizing each sample to a new substrate, and Shows a method of manufacturing the DNA probe chip obtained.

Figure 3a is a conceptual diagram showing the analysis results by the analysis method of the gene expression aspect of the present invention, Figure 3b is a fluorescence micrograph showing the gene expression pattern analyzed by the gene expression pattern analysis method using the DNA chip of the present invention. 3c is a photograph showing the results of the assay through the Northern blot analysis.

The present invention relates to a method for producing a DNA chip for analyzing a gene from a transgenic plant, a DNA chip produced by the method and an analysis method using the same.

DNA sequence information is rapidly increasing due to the development of molecular biology technology and the 'human genome project', and the speed is further accelerated through the development of analysis tools for sequences. One of the major challenges of modern molecular biology is to develop fast, low-cost techniques for applying new genetic information to diagnosis. A recent development of DNA diagnostic tools and genetic analysis tools is a DNA chip in which DNA molecules are densely arranged on a two-dimensional surface. DNA chips are technologies that attach thousands of tens of thousands of DNA to very small metals or glass surfaces at high density, and analyze genes that hybridize with these DNA at high speed. This technique was derived by requiring large-scale gene expression analysis in organisms when the entire genetic map of organisms such as E. coli is revealed and the sequence of up to 100,000 human genes is revealed. Traditional molecular biology methods, such as southern blot analysis and northern blot analysis, are derived from the reasons that such large-scale genetic analysis is impossible due to time or cost limitations. In addition, due to the development of mechanical automation, electronic control technology, etc. that can apply the related technology, a technology that can integrate hundreds of thousands of DNA in a very small space has been possible.

DNA chip is composed of cDNA chip that has 100bp or more genes (full-length open leading frame or EST) and DNA synthesized by chemical reaction with less than 100bp base according to the characteristics and size of genetic material immobilized on the substrate. It can be divided into oligonucleotide chips. The oligonucleotide chip accumulates DNA probes consisting of 20 to 50 nucleotides, hybridizes probe DNA and sample DNA consisting of a combination of four nucleotides (A, G, C, and T), and sequence them. DNA sequence and mutated sequences are analyzed by changing the signal size according to the degree of agreement. Complementary DNA microarray chip (cDNA microarray chip) integrates the known open reading frame (ORF) into the chip, and synthesizes cDNA with reverse transcriptase in vivo and hybridizes with ORF to change the signal size according to its expression level. By analyzing the expression level of a particular gene. DNA chips are arranged on the chip surface by synthesizing DNA molecules directly on the surface of the chip or by immobilizing pre-synthesized DNA molecules on the surface.

DNA chips may be classified into pin microarray chips, inkjet microarray chips, photolithograph chips, and electronic array DNA chips according to specific manufacturing methods. Pin micro-chip substrate in Stanford 1995 (Stanford, Dr. Pat. Brown ) was developed at the University of 2-3000 genes within 1cm ² chip to be implanted on a chip and a cDNA nucleotide pin (pin) raised pre-production Integration is possible (Schena M., et al., Science, 270 (5253): 467-470, 1995). Specifically, it consists of three parts: a substrate (poly-L-lysine is treated on the glass surface for binding to a gene), probe DNA (DNA on 96-well amplified by PCR and fixed on the substrate), and pins are mounted. Robotic print head (robot print head) to move the DNA from the well to the glass substrate to secure. The photoetch DNA chip is an oligonucleotide arrayed on a substrate using a photoetch technique commonly used in semiconductor processes (US Pat. No. 5,143,854).

However, the method of manufacturing the DNA chip as described above requires a mask having a respective pattern for each production of the DNA chip, which is complicated in the mass production process and requires expensive equipment so that the manufacturing cost is high and the washing and masking are performed for each process. There is a problem that the alignment process is required.

The inkjet microchip DNA chip is a method of using a cartridge of the same principle as the cartridge used to eject the dye solution in the inkjet printer instead of the pins used in the pin microsubstrate chip. Each cartridge contains a fixed gene. Genes are sprinkled onto a solid by electric force, and there are thermal, solenoid, and piezoelectric printing methods. The advantage of this technology is that the gene can be sprinkled without touching the surface of the chip electrically, so that it can produce a large number of chips with the quantitative gene attached, but there is a drawback that occurs when the sample is injected and the injection device is operated. have. In addition, when the piezoelectric printing method is used, there is a method of preparing an oligonucleotide on the surface by electrically releasing one of four bases (US Patent No. 5,474,796).

As a result, the DNA chips have a disadvantage in that the manufacturing process is complicated and the manufacturing cost is high and there is a problem in mass production, and the expensive DNA chips are used in large numbers when analyzing multiple genes.

Compared with the existing typical gene analysis methods such as Southern blot analysis, Northern blot analysis, mutation detection and DNA sequencing, the genetic analysis method using the DNA chip can search at least several hundred genes in a short time. By using the same solid substrate, it is possible to attach a very small amount of genetic material at a high density and simultaneously search a large number of genes. The detection of gene expression has the advantage that the cDNA chip or oligonucleotide chip is superior to all existing methods. In addition, oligonucleotide chips can also be used for mutation detection, making a significant contribution to mutation detection, disease diagnosis or gene expression blueprints. There are various methods of analyzing the existing mutant genes. A commonly used mutant analysis method is DNA sequencing, and restriction fragment length polymorphism (RFLP) is a restriction enzyme that selectively cuts specific sequencing. Using the gel electrophoresis to separate and analyze the cut pieces to know the pattern. If the restriction enzyme cuts the position of the mutated base, the size of the cut pieces will change, so you can see the mutation of the gene. In addition, PCR techniques using primers suitable for the mutant portion, ligase (ligase) method (using two oligomers to ligated the ligated part, combined with ligase, if not ligated, if not ligated) Southern blot analysis The method of detecting by oligomer probe by Northern blot analysis and the use of a DNA chip can be various methods (Mathew CG., Methods in Molecular Biology Vol. 9, Humana Press, NJ, 1991).

However, these conventional methods have attracted interest in methods using DNA chips because only one mutation analysis can be performed at a time or complex gel electrophoresis analysis must be involved.

In conclusion, cDNA chip is composed of genes consisting of at least 100 bp base, which is mainly used for research purposes such as the expression of intracellular protein expression and gene function. It is possible to find out the difference in genetic information due to mutations, etc. is difficult. In addition, the oligonucleotide chip is an oligonucleotide of less than 100 bp attached to the substrate, unlike the cDNA chip has the advantage of distinguishing the difference between the single base, it is possible to search for mutations, but the gene bound to the oligonucleotide Because you don't know which gene is, you only need to amplify a specific region using PCR, and there is a limitation that the gene region that can be searched on one chip is limited. When analyzing using a DNA chip, a large number of genes are analyzed at one time. This is difficult and does not only serve the purpose of genetic analysis, but also the use of the DNA chip for further use.

The expression patterns of all genes in an organism affect the expression of other genes, and the overall biological response is caused by systemic biologic respose related to gene correlation.

Stilbene synthase, also called resveratrol synthase, is a kind of polyketide synthases (hereinafter referred to as PKS) and is called polyketide by their expression. To produce secondary metabolites. Recently, many pharmacological activities of these have been revealed, and have been used as antibiotics, immunopotentiators, anticancer agents, and antimicrobial agents (Hutchinson, Curr. Opin. Microbiol., 1 (3): 319-329, 1998). . PKS is usually divided into three types, and enzymes belonging to the third type belong to the group of chalcone synthase (CHS) and stilbene synthase (STS, subunits 40-45kDa) and are expressed in plants. To form phenolic compounds through decarboxylative condensation and cyclization steps (Martin CR, Int. Rev. Cytol., 147: 233-283, 1993). From one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA, CHS and STS form chalcone and resveratrol, respectively.

Resveratrol ( trans -3,5,4'-trihydroxystilbene) is expressed in limited interrelated plant species such as Morus alba , grapes ( Vitis vinifera ) and peanuts ( A. hypogaea ) and is called "French paradox.""Frenchparadox" is reported to have a much lower rate of death from aromite atherosclerosis than other fats, although people who drink red wine frequently in some parts of France consume much fat. It is a substance that concentrates consumption and attention. Resveratrol is known to play a role in preventing skin cancer in mice and proliferation of human leukemia cells on the basis of potent antibacterial and antioxidant activity (Jang M., et al., Science , 275). (5297): 218-220, 1997). It is also known to induce platelet aggregation in mice, inhibit the oxidation of low density lipoproteins in humans and protect the liver from lipid peroxidation (Palomino O., et al., J. Chromatogr. A. , 870 (1-2): 449-451, 2000). These substances are involved in redox sensitive cell signaling pathways on the basis of potent antioxidant activity (Bertelli AAE, Pharmaceut. Biol., 36: 44-52, 1998). Effective removal of reactive oxygen species (ROS) prevents oxidative DNA damage of the kidneys induced by KBrO ₃ (Cadenas and Barja, Free Radic. Biol. Med., 26 (11-12): 1531-1537, 1999), substances that can help successful organ transplantation by removing peroxyl radicals from rejection of isolated heart transplants in rats (Ray PS, et al., Free Radic. Biol. Med. , 27 (1-2): 160-169, 1999). Resveratrol also inhibits apoptosis of K562 cells by directly inhibiting lipoxygenase or cyclooxygenase by regulating the check point of the cell cycle. (MacCarrone M., et al., Eur. J. Biochem., 265 (1): 27-34, 1999). Stilbene and stilbene derivatives such as resveratrol in plants are known to act as phytoalexins as a defense against microbial infection.

Accordingly, the present inventors have confirmed the expression of the gene encoding the stilbene synthase from the refractory plant transformed by the gene encoding the stilbene synthase encoding the resveratrol and related derivative synthesis, and in the plant expressing the gene The present invention was completed by making a DNA chip capable of confirming changes in expression patterns of other related genes.

An object of the present invention is to provide a gene analysis method using a DNA chip that can confirm the expression of the gene and the change of expression of other genes in the same plant from a sulfur-like plant transformed with a specific gene.

In order to achieve the above object, the present invention provides a method for producing a DNA chip for genetic analysis of a plant transformed with a specific gene.

More specifically, the method for producing a DNA chip of the present invention

(Iii) preparing a DNA capture chip by arranging DNA complementary to the nucleic acid to be analyzed on a substrate;

(Ii) a second step of preparing a sample solution containing the nucleic acid to be analyzed;

(Iii) a third step of hybridizing the nucleic acids to be analyzed by adding the sample solution of the second step to the DNA capture chip of the first step, and removing the nonspecifically hybridized nucleic acid molecules; And

(Iii) a fourth step of transferring the assay nucleic acids complementarily bound to the corresponding DNA to a new substrate.

At this time, the DNA complementary to the nucleic acid of interest is not particularly limited, but preferably includes a gene encoding the stilbene synthase derived from peanut ( Arachis hypogaea L.) (SEQ ID NO: 1), The subject nucleic acid is not particularly limited, but is preferably a nucleic acid of a transformed plant including DNA or RNA, wherein the transformed plant introduces a gene encoding a stilbene synthase, although not particularly limited thereto. It is preferred that it is one transgenic sulphurous plant. On the other hand, DNA complementary to the nucleic acid to be analyzed is not particularly limited, but is preferably DNA amplified by a precursor capable of amplifying a gene fragment encoding an active site of stilbene synthase. In the method for producing a DNA chip for genetic analysis of the present invention, it is more preferable to repeat the third step and the fourth step further, the DNA chip is arranged in a specific position a plurality of gene sites including the active site, It is desirable that two analyte cDNAs compete with the probe to obtain genetic information of one or more genes in the sample at the same time. In addition, the sample solution in the method for producing a DNA chip for genetic analysis of the present invention is not particularly limited thereto, but preferably comprises a cDNA consisting of a sequence capable of specifically binding to the nucleic acid to be analyzed and labeled with a fluorescent material, In this case, the fluorescent material is not particularly limited thereto, but is preferably Cy3 ^™ or Cy5 ^™ .

In addition, the present invention provides a DNA chip produced by the above method.

In the DNA chip fabrication process, if the above steps are repeatedly performed, a plurality of DNA probe chips in which nucleic acids of the same sequence are arranged may be obtained.

In addition, by washing the DNA chip after the fourth step and adding a sample solution again, the third and fourth steps can be obtained to obtain a DNA probe chip arranged nucleic acid of the same sequence.

The DNA chip of the present invention arranges a plurality of gene sites including an active site at a specific position, and at least one cDNA of two analytes has competitive binding with a probe, thereby providing at least one present in the sample. Genetic information of genes can be obtained simultaneously (see FIG. 1).

1 is a conceptual diagram showing in detail a process of analyzing a resveratrol synthetic gene using a DNA chip from a sulfur-based plant transduced with a resveratrol synthetic gene.

The present invention also provides a method for analyzing a stilbene synthetic gene using the DNA chip from a plant transformed with a stilbene synthetic gene.

In addition, the present invention provides a method for analyzing gene expression patterns between a plant transformed with a foreign gene and a natural plant.

More specifically, the gene expression pattern analysis method of the present invention

(Iii) a first step of preparing a DNA chip in which two nucleotide groups of two analytes have been deposited;

(Ii) extracting mRNA from the analyte;

(Iii) a third step of preparing probe DNA by labeling different fluorescent substances on the mRNA;

(Iii) a fourth step of mixing the probe DNA with the DNA chip of the first step and washing and removing the unmixed probe DNA;

(Iii) a fifth step of detecting fluorescence of the DNA chip of the fourth step with a fluorescence analyzer.

Hereinafter, the present invention will be described in detail.

Referring to the process of manufacturing the DNA chip of the present invention in more detail,

(Iii) preparing a DNA capture chip by arranging DNA complementary to the nucleic acid to be analyzed on a substrate;

(Ii) a second step of preparing a sample solution containing the nucleic acid to be analyzed;

(Iii) a third step of hybridizing the nucleic acids to be analyzed by adding the sample solution of the second step to the DNA capture chip of the first step, and removing the nonspecifically hybridized nucleic acid molecules; And

(Iii) a fourth step of transferring the assay nucleic acids complementarily bound to the corresponding DNA to a new substrate.

The nucleic acid to be analyzed in the first step is not particularly limited thereto, but is preferably cDNA prepared by reverse transcription of mRNA extracted from the whole plant transformed with a foreign gene, and more preferably, a trait introduced with stilbene synthase. CDNAs prepared from the conversion of the sulfurized plant and the native type of the sulfurized plant. DNA complementary to the nucleic acid of interest is not particularly limited, but is preferably a PCR product amplified from a precursor capable of amplifying a gene encoding an active site of a specific enzyme, more preferably stilbene synthesis. It is a PCR product amplified from a precursor capable of amplifying a gene encoding an active site of an enzyme.

In addition, the analysis method of the gene using the DNA chip of the present invention

(Iii) a first step of preparing a DNA chip in which a group of nucleotides is deposited;

(Ii) a second step of extracting mRNA from two different analytes;

(Iii) a third step of preparing probe DNA by labeling different fluorescent substances on the mRNA;

(Iii) a fourth step of mixing the cDNA with the DNA chip of the first step and washing and removing the unmixed cDNA;

(Iii) a fifth step of detecting fluorescence of the DNA chip of the fourth step with a fluorescence analyzer.

In this case, the analyte is not particularly limited thereto, but is preferably a plant transformed with a foreign gene and a natural plant, and the plant is not particularly limited thereto but is preferably sulfur, and the foreign gene is particularly limited thereto. Although not preferred, it is preferably a gene encoding a stilbene synthase, and the nucleotide group is not particularly limited thereto, but is preferably selected from the group consisting of cDNA, fragments of cDNA, and oligonucleotides expressible from the analyte. , More preferably, positive control and / or negative control nucleotides. The probe DNA is not particularly limited thereto, but is preferably cDNA prepared from mRNA extracted from an analyte, and the fluorescent material is not particularly limited thereto, but Cy3 ^™ and Cy5 ^™ are preferably used, respectively. Fluorescence detection can be performed by known methods (Nature Genetics Supplement, vol. 21, p10, 1999).

In addition, the DNA chip subjected to the fifth step is washed, and a plurality of identical DNA probe chips to which the nucleic acid to be analyzed are combined are prepared through the fourth and fifth steps, and a different DNA probe set is prepared. By hybridizing to the DNA probe chip, it is also possible to analyze the genetic information of each gene having a different gene or a different mutant base.

The DNA chip of the present invention is a DNA chip in which the DNA to be analyzed is immobilized on a substrate. Thus, if there are multiple DNA in the sample, each of them binds to the specific position of the phase and then is fixed again.

In the present invention, a substrate for manufacturing a DNA probe chip may be purchased and used commercially, and may be manufactured with multiple lysine-coated slide glasses (S7444, Metunami, Osaka, Japan) and the like.

As used herein, the term "probe DNA" refers to a DNA for probe having a base sequence that can be complementarily bound to an assay nucleic acid.

In addition, the genetic analysis method using the DNA chip of the present invention is labeled with a reporter molecule on the DNA or DNA to be fixed on the DNA chip, the fluorescent signal or radiation based on complementary binding of the probe DNA on the DNA chip and the DNA to be analyzed. Can be analyzed to obtain genetic information.

There is a need for probes labeled with fluorescent molecules having different fluorescence properties for each gene, but the number of molecules that can be classified as fluorescence properties is limited to a few dozen. In the present invention, since a plurality of genes are arranged in a two-dimensional plane and the information of the genes can be distinguished according to the arrangement position, a probe of a known nucleotide sequence capable of binding to the gene with a limited number of fluorescent molecules can be prepared and distinguished from each other. By combining fluorescent molecules and combining them with the corresponding probes for multiple genes at the same time to analyze the fluorescence wavelength at each position, multiple genes can be analyzed even with limited fluorescent molecules. For example, for two comparison groups consisting of transformed plants and natural-type plants in which 100 resveratrol synthetic genes were introduced into 100 genes, respectively, two fluorescent molecules were coupled to probe DNA corresponding to the mutation ( In this case, two fluorescent molecules are used for one gene, but even if 100 genes are used, the same fluorescent molecules can be used for each one, so 200 fluorescent molecules are actually used.) A total of 400 probes are mixed with DNA probe chips. Since the gene is combined with the probe corresponding to the mutation for each gene, analyzing the fluorescence signal shows the nucleotide sequence of the mutation region of each gene from the sample. You can get information.

The fluorescent molecules to be bound to DNA are not particularly limited, but it is preferable to use Cy3 ^TM or Cy5 ^TM . The method of binding these fluorescent molecules is, for example, a method of synthesizing oligomers, a method using an enzyme, or the like. There are a variety of methods commonly known in the art.

The present invention also provides an analysis method for analyzing the mutant gene using the DNA chip.

More specifically, in the hybridization of the DNA chip manufacturing process, the mutant gene introduced by transformation by hybridizing probes labeled with fluorescent molecules having different fluorescence wavelengths to DNA fragments that can hybridize to normal and abnormal genes, respectively. It is an analysis method that can distinguish between and wild type genes.

In addition, the present invention can prepare a plurality of identical DNA probe chip to which the nucleic acid to be analyzed, and hybridize different DNA probe sets to each DNA probe chip to analyze the genetic information of the mutation position.

Probes of specific genes to be used can be prepared with a general oligonucleotide synthesizer, and commercially available oligonucleotides can be purchased and used.

The present invention utilizes the properties of hybridization and denaturation of nucleic acids, and thus, a chip having a gene derived from a sample obtained by transferring a gene selectively positioned such as a DNA chip through denaturation onto a new substrate, that is, By using the probe chip, and again using Southern blot analysis or Northern blot analysis to determine the nucleotide sequence information of the region where the mutation occurs by using the oligonucleotide as a probe, it is possible to read whether or not the nucleotide sequence from the sample.

Precursors for the production of each oligonucleotide probe are designed to have similar Tm and hybridize to the same DNA sequence because the probe is about 25bp long, but the DNA sequence is hybridized at different sites of the similar gene, resulting in fluorescence image Possible interference occurs. In order to reduce or eliminate such interference, if an oligonucleotide having a DNA sequence that is bound by a few bp to the interference causing portion is hybridized without adding a fluorescent molecule, an oligonucleotide probe that causes interference is hybridized. By blocking the position, you can prevent the wrong signal coming out.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Analysis of Stilbene Synthetic Gene Using DNA Chip

DNA chip of the present invention using a prosite program (PROSITE program, http://us.expasy.org/) using a precursor prepared by identifying a clear active recognition site (active core part) that has bioinformatically active ( PCR is performed using a primer), and the DNA product is fixed by a two-dimensional array of DNA having a specific sequence of a specific gene region, thereby producing a general DNA chip (see FIG. 2). .

2 is a method for retrieving amino acid sequences for genes capable of synthesizing resveratrol-type compounds using a PROSITE program (http://us.expasy.org/) and determining sequences Method and amplification of resveratrol synthase and key enzymes of other genes using PCR, and amplified PCR products are deposited on a substrate (glass), and each sample is hybridized and transferred to a new substrate. It is a conceptual diagram which shows the method of manufacturing the DNA probe chip obtained by this.

Nucleic acid in the sample solution used for the DNA chip is a messenger RNA capable of complementary binding to each other on the DNA chip is used, the fluorescent molecules are labeled and hybridized, applied to the DNA chip and analyzed by obtaining a fluorescent image.

The fabrication and gene analysis methods of specific DNA chips are described below.

Example 1-1 Preparation of Precursor

Genes and primer sets selected for the production of DNA chips are as follows. First, each gene was identified using a DNA prosite program to identify a clear bioinformatic activity. Selected genes and primers were RS3-5 '(SEQ ID NO: 2: 5') selected from the gene encoding the stilbene synthase of peanut ( Arachis hypogaea L.) (Genebank Accession No. 166476, SEQ ID NO: 1). -catcc atg gtgtctgtgagtgaattc-3 ') and RS3-3' (SEQ ID NO: 3'5'-gtattatatggccatgctgcggag-3 '), from chalcone synthase (CHS, Accession No. BAA31259, SEQ ID NO: 4) isolated from grapes ( Vitis vinifera ) Genes isolated by selected CHS-5 '(SEQ ID NO: 5'-cgtttctatatcaatcataatat-3') and CHS-3 '(SEQ ID NO: 5'-ttgtgtaagttgtttaaataa-3'), Vibrio vulnificus CMCP6 DtdS-A1 (SEQ ID NO: 8: 5'-ggtcacaaaaaaccatcccag-3 ') selected from the threonine dehydrogenase gene (threonine dehyrogenase, dtdS, Genebank Accession No. 27359410, SEQ ID NO: 7) and dtdS-A2 (SEQ ID NO: 9: 5'-) catttcacggccatagatacc-3 '). Meanwhile, as a positive control, α-tubulin 5 '(SEQ ID NO: 11: 5) selected from Arabidopsis thaliana alpha-tubulin 6-chain gene (α-tubulin 6-chain, Genebank Accession No. 17473672, SEQ ID NO: 10) '-ggaaagtacatggcttgctgtttgatg-3') and oligo d (T) ₁₆ were used, and the negative control group was the nicotinic acetylcholine receptor beta subunit, nAchRE, Genebank Accession No. 191603 in Mus musculus . NAChRe-5 '(SEQ ID NO: 13: 5'-aggcgcgcactgacaccactaag-3') and nAChRe-3 '(SEQ ID NO: 14: 5'-gactgctgtgggtttcacccag-3') selected from SEQ ID NO: 12 were used. The alpha-tubulin gene was conserved to a high degree in plants, so it is normal to be expressed at the same level in both transformed and natural plants, so it was used as a positive control group, and the nicotinic acetylcholine receptor beta subunit in mice was used only in animals. Since it is expressed, it was used as a negative control. The precursors were all synthesized using an oligonucleotide synthesizer (ABI 394 oligonucleotide synthesizer, Applied Biosystems Inc., USA).

Example 1-2 PCR Reaction and Arrangement of PCR Product to Substrate

PCR reactions were performed using the precursors prepared in Example 1-1. PCR reaction conditions were PCR products (PCR Express, HYBRID, USA) capable of carrying out a commercial polymerase chain reaction (CRT Express, HYBRID, USA) was used, cDNA synthesis kit (Stratagene) by extracting mRNA from the living body of each gene origin , USA) was used to prepare cDNA and PCR as a template. Looking at the specific PCR reaction conditions, the reaction mixture was prepared to mix the template cDNA 1 ng, Taq DNA polymerase 5U, 1 mM each of dNTP, 100 pmol each of the primer, and distilled water to a total volume of 50 μl. The DNA fragments were denatured by reacting for about 1 minute at 94 ° C, 1 minute at 59 ° C, and 1 minute at 72 ° C for 35 minutes to amplify the DNA fragments. Then, the reaction was finally reacted again at 72 ° C. for 10 minutes to obtain a PCR product. The obtained PCR product was electrophoresed on 1% agarose gel and stained with ethidium bromide and assayed under ultraviolet light. The PCR product (100 ng / ml) obtained through the above process was coated on a glass substrate coated with 384 drops of poly-L-lysine (SpotBot ^TM Complete Desktop Manufacturing System, Telechem, USA) was used to drop by dropping by 0.5 nl. At this time, the distance between the droplets was set to 280 ㎛.

The dipped glass plate was supplied with water using sterile steam and dried again at 100 ° C. for 3 seconds. The glass plate was placed in a glass plate mold, and 15 ml of 1 M sodium borate (pH 8.0), 5.5 g of succinic anhydride and 335 ml of 1-methyl-2-pyrrolidone (1-methyl Absorb the mixed solution of -2-pyrrolidone) and shake gently for 15 minutes. The glass plate mold was transferred to a container containing boiling water and allowed to stand for 2 minutes, and then centrifuged at 2500 g for 30 seconds using a microarray high speed mini centrifuge (Telechem, USA).

Example 1-3 Preparation of Sample Solution Containing Analysis Target Nucleic Acid

Phosphotransfected plants incorporating the stilbene synthetase as an analyte were subjected to the T-bounds of pMG643, an agrobacterium transformation vector, with the corresponding stilbene synthase (RS3, AF227963) isolated from a pod of peanut ( Arachis hypogaea L.). A binary vector system consisting of a plasmid and a helper plasmid prepared by inserting in an L-boundary was constructed, and transformed into ABA bacterium ( Agrobacterium tumefeciens ) type LBA4404 with the constructed plasmid, and then transformed into the transformed Agrobacterium. After inoculation and co-cultivation of Rhemannia glutinosa L. plants to be converted, extra bacteria were removed, plants were re-differentiated and plants expressed by the enzymes were used. It was confirmed through. Nucleic acid extraction was performed by using a phenol extraction method, and purified by mRNA purification kit (Oligotex-dT ₃₀ , Takara, Japan).

The nucleic acids extracted from each analyte were reverse transcribed into Cy3 dUTP and Cy5 dUTP, respectively. 30 μl total, poly (A) ⁺¹ μg RNA, 6 μg 18 oligo-dT (oligo-dT), 10 mM DTT, 500 μM dATP, dCTP and dGTP, 200 μM dTTP, 100 μM Cy3- dUTP or Cy5-dUTP and reverse transcriptase (400 units, SuperScript II reverse transcriptase, Life Technologies, USA) were used. After the reaction was mixed, 15 μl of 0.1 M NaOH and 1.5 μl of 20 mM EDTA were added and incubated at 70 ° C. for 10 minutes, and then 15 μl of 0.1 M HCl was added again. Transfer the treated sample to a microcondenser (Microcon 30 microconcentrator, Amicon, USA) and add 400 μl of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), then centrifuge for 5-10 minutes. The final volume was adjusted to 12 μl by separation.

2 μl of yeast tRNA (10 μg / μl) and 2 μl of poly-d (A) _12-18 (1 μg / μl, Amersham Pharmacia, Sweden) were added to the reaction, 3.4 μl of 20 × SSC and 10% (v / w) 0.6 [mu] l of SDS was added and incubated for 1 minute in a water bath at 100 DEG C, and then left at room temperature for 30 minutes.

The fluorescently labeled cDNA probe was denatured by heating at 95 ° C. for 2 minutes, centrifuged at 15,000 rpm for 25 minutes at 25 ° C. after cooling at room temperature, and then placed in a constant temperature zone to prevent precipitation. after that. After centrifugation, the supernatant was separated to cover the cover glass, and a pipette was inserted between the cover glass and the DNA chip. The cover glass used has a spacer having a thickness of about 20 μm at the edge of the cover glass, and there is a space for the hybridization solution with the DNA chip. By using this, the solution can be uniformly diffused on the DNA chip to obtain a good hybridization image.

The mixture was competitively hybridized on a DNA chip, and the basic data was the difference in the amount of mRNA expression between samples, that is, the difference in the amount of each gene. The fluorescent substrate used is dUTP, to which Cy5 ^™ and Cy ^™ are bound, respectively, and is labeled on the cDNA with almost the same efficiency. Since the fluorescent material is different from the excitation wavelength and the emission wavelength, each fluorescent intensity can be measured.

Example 1-4 Hybridization Process

The DNA chip was prepared by adding the above-described sample containing the nucleic acid of interest to the glass plate in which the PCR product used as the probe was dipped.

The DNA of the DNA chip was placed in a square Petri dish with the dipped side up and fixed with tape. After sealing the square Petri dish fixing the DNA chip, a paper towel (KimWipes, South Korea) soaked in water was placed under the airtight container to maintain humidity in the container. Insulate the sealed container in a 65 ° C constant temperature water bath for 12-16 hours, and set two sets of containers with 2 × SSC / 0.2% SDS solution to 55 ° C and one set of 2 × SSC / 0.2% SDS solution to 65 ° C. Preheated The DNA chip was placed in a 2 × SSC / 0.2% SDS solution and the cover glass was removed while surrounding so that the spot where the DNA was deposited was not damaged. The DNA chip was washed with stirring at 55 ° C., 2 × SSC / 0.2% SDS solution for 5 minutes. Wash at 65 ° C., 2 × SSC / 0.2% SDS solution for 5 minutes and at room temperature with 0.05 × SSC solution. DNA chips were dried by low speed centrifugation at 1000 rpm for about 2 minutes and then completely removed with compressed N ₂ gas.

Example 1-5 Genetic Analysis of DNA Chips

The DNA chip hybridized in Example 1-4 was analyzed by fluorescence images in the following manner. Images were analyzed with a model array WoRxe Biochip reader (BMS, USA) with a CCD camera and the resolution of each phosphor was adjusted to 10 μm per pixel. Each two signal strengths were normalized to two channels. Data analysis was analyzed using Biochip analysis software (WoRxe software, BMS, USA). Because of the competitive hybridization, the amount of each mRNA is not an absolute amount, but a relative amount of Samples A and B. Fluorescence intensities of Cy3 ^TM and Cy5 ^TM were obtained as image data using a DNA chip analyzer, and then quantified using the biochip analysis software. . The expression ratio (mRNA amount) of each gene was analyzed by analysis software, and the results were obtained by a svarrer plot, pie graph form, image overlap, and the like. The normalization of the signal intensity of Cy3 ^TM and Cy5 ^TM is expected to be small in the expression level between cells, and thus, tubulin and negative control, which are housekeeping genes used as a control in gene expression analysis, such as the Northern blot analysis. NAChRE (mouse nicotinic acetylcholine receptor beta subunit) was used. The numerical results were quantified by quantifying the amount expressed in the transgenic plant and the amount expressed in the natural plant, and then quantifying the relative ratio of the signal observed in the transgenic plant / signal observed in the natural plant ( 3a and 3b). On the other hand, errors of data obtained from DNA chips were assayed by general Northern blot analysis (FIG. 3C).

Figure 3a is a conceptual diagram showing the analysis results by the analysis method of the gene expression aspect of the present invention, Figure 3b is a fluorescence micrograph showing the gene expression pattern analyzed by the gene expression pattern analysis method using the DNA chip of the present invention. 3c is a photograph showing the results of the assay through the Northern blot analysis. At this time, in FIG. 3B, CHS means chalcone synthase isolated from grapes, dtdS means threonine dehydrogenase gene (threonine dehyrogenase) of Vibrio vulnificus CMCP6, and RS3 is still derived from peanut. Refers to a gene encoding a ben synthase (stilbene synthase), nAChRE refers to the nicotinic acetylcholine receptor beta subunit of mice, α-tubulin is alpha-tubulin 6 of the Arabidopsis Chain gene.

As shown in FIG. 3B, the PCR product amplified with the RS3 primer showed a signal intensity of about 15 to 18 in a sulfur-like plant transformed with a gene encoding a stilbene synthase, and one point or more in a natural plant. It was assayed below. As expected, the positive control alpha-tubulin was expressed in both transformed and native plants and showed a yellow signal, while the negative control nAChRE did not detect a signal. Of particular note, the CHS gene was not detected in non-transformed native plants, while strong signals were detected in plants transformed with genes encoding stilbene synthase. As can be seen from the above experimental results, when using the DNA chip for genetic analysis of the present invention, it can be seen that in addition to the introduced genes in the transformed plant, the expression patterns of other genes showing systemic biological activity can be analyzed. Could.

As claimed and demonstrated above, the analytical method for analyzing gene expression patterns from the transgenic plant of the present invention expresses the gene by using the active site of any gene as a probe in the organism. It is possible to quickly test whether or not the expression patterns of a plurality of genes can be compared using a transgenic plant into which a gene is introduced, and thus the expression pattern of other genes by expression of the gene in a plant into which the modified gene is inserted. It can be useful for analysis.

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Claims (18)

(Iii) Amplify a gene fragment encoding the active site of stilbene synthase, a DNA capable of complementarily binding to DNA or RNA extracted from a transgenic plant that has introduced a gene encoding a stilbene synthase (SEQ ID NO: 1). A first step of preparing a DNA capture chip by arranging a nucleic acid comprising a gene (SEQ ID NO: 1) encoding a stilbene synthase amplified by a precursor capable of ; (Ii) a second step of preparing an analyte nucleic acid sample solution containing DNA or RNA extracted from a transfected bovine plant and a wild type yolk introduced with a gene encoding a stilbene synthase ; (Iii) a third step of hybridizing the nucleic acids to be analyzed by adding the sample solution of the second step to the DNA capture chip of the first step, and removing the nonspecifically hybridized nucleic acid molecules; And (Iii) a method for producing a DNA chip for gene analysis, comprising a fourth step of moving the analyte nucleic acids complementarily bound to corresponding DNA to a new substrate. delete delete delete delete The method according to claim 1, wherein the third and fourth steps are additionally repeated. The method of claim 1, wherein the DNA chip is arranged to arrange a plurality of gene regions, including the active site in a specific position, the two cDNA to be competitively coupled with the probe to obtain the genetic information of one or more genes present in the sample at the same time Characterized in the manufacturing method. The method of claim 1, wherein the sample solution is composed of a sequence that can specifically bind to the nucleic acid to be analyzed and is characterized in that it comprises a cDNA labeled with a fluorescent material. The method of claim 8, wherein the fluorescent material is Cy3 ^™ or Cy5 ^™ . A DNA chip for gene analysis prepared by the method of any one of claims 1 and 6 to 9. (Iii) a DNA chip in which a plurality of nucleotides containing a gene encoding a stilbene synthase (SEQ ID NO: 1) is selected from the group consisting of cDNA, fragments of cDNA including PCR products, and oligonucleotides Preparing a first step; ( Ii ) extracting mRNA from rhemannia glutinosa L. and wild type yolk transformed with a gene encoding a stilbene synthase (SEQ ID NO: 1), respectively ; (Iii) a third step of preparing probe DNA by labeling different fluorescent substances on the mRNA; (Iii) a fourth step of mixing the probe DNA with the DNA chip of the first step and washing and removing the unmixed probe DNA; (Iii) Gene analysis method using a DNA chip comprising a fifth step of detecting the fluorescence of the fourth step of the DNA chip with a fluorescence spectrometer. delete delete delete delete 12. The assay of claim 11 wherein the phosphors are Cy3 ^™ and Cy5 ^™ , respectively. 12. The method of claim 11, further comprising the step of calculating the ratio of fluorescent signals of different fluorescent materials. 12. The analysis method according to claim 11, wherein the fourth and fifth steps are additionally repeated.

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