KR20130037785A - Investigation of radio marker gene at4g37990-aromatic alcohol:nadp+ oxidoreductase as a radiomarker gene and transgenic indicator plant using the gene - Google Patents

Abstract

The present invention relates to a radiolabeled gene and a transgenic marker plant using the same. More specifically, after irradiation of gamma rays of various doses to the trophic Arabidopsis plants, the microarray technique is used to specifically expose gamma rays. At4g37990 (aromatic alcohol: NADP + oxidoreductase) having increased expression is selected, and then, kits and transgenic plants using the gene can be usefully used to confirm gamma-ray exposure.

Description

Investigation of Radio marker gene At4g37990-aromatic alcohol: NADP + oxidoreductase as a Radiomarker gene and transgenic indicator plant using the radioactive marker plant using the

The present invention relates to a search for a novel radiolabeled gene and a transformed indicator plant using the same. More specifically, the present invention relates to At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene as a radiolabeled gene and to confirm radiation exposure using the radiolabeled gene. Kits, RNAi vector construction using the radiolabeled gene, and production of a transgenic plant with the vector and a method for confirming radiation exposure using the transgenic plant.

Exposure to radiation, the source of physical stressors, can cause DNA damage at the cellular and molecular levels of the organism, and even to severe cell death (Howley et al., Int. J. Radiat . Biol . 78, 1095-1102, (2002)). In particular, genetic abnormalities, such as double strand breaks in DNA, can be fatal enough to cause biological death (Sachs et al., Int . J. Radiat . Biol . 72, 351-374, (1997). As a defense against this, organisms can operate damaged DNA repair mechanisms through their own repair mechanisms, such as nonhomologous end joining, homologous recombination, and single strand annealing. (Kobayashi et al., J. Radiat. Res . 49: 93-103, (2008)). In addition, free radicals generated in the mitochondrial electron transfer system in the organism by radiation can cause significant damage to cells or tissues. This array of procedures is being conducted to study the mechanism of the plant corresponding to the oxidative stress response in plants (Nagata et al., J. Agric . Food Chem . 51,2992-2999, (2003).

Until now, research on plant responsiveness to radiation has been conducted mainly on the study of DNA damage and repair mechanisms or antioxidant related mechanisms, and studies on genes that respond to radiation and related signal transduction have been conducted in animal models. It has been limited (Amundson et al., Radiat. Res . 156, 657-661, (2001)).

Arabidopsis thaliana is the scientific name of Arabidopsis as a biennial cruciferous is thaliana . When the size of the plant grows up, it is 15 ~ 35㎝. Root leaves gathered and inverted lanceolate, stem leaves without lanceolate in lanceolate. Small white flowers bloom from the end of stem in April-May. The fruit is a nut. It grows in the fields of Gyeonggi-do and Jeollanam-do provinces in Korea. The first generation from germination to the next seed is short, about six weeks, and chemicals can induce a variety of mutants. In addition, the small size of the plant allows for easy cultivation even in small spaces and the small genome size. For this reason, it is widely used as a model plant for plant research. At the end of 2000, the nucleotide sequence of the entire genome (approximately 125 million) was almost completely deciphered and 2,400 genes were found. Among the genes, many common to the genes of rice and wheat, the monocotyledonous plants grown, are used to decode the genes. do.

Therefore, the present inventors endeavored to develop an indicator plant capable of detecting gamma-ray exposure in a plant ecosystem, and as a result, a plant gene expression profile (profiling) that responds to radiation doses by using microarray technology is produced. After preparing and selecting the At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene whose expression is increased by radiation, the radiation reactivity of the gene is examined, and a kit for confirming radiation irradiation using the gene is prepared, or the gene The present invention was completed by uncovering the possibility of detecting the exposure to plant ecosystem radiation by preparing a transformant having introduced.

An object of the present invention is a knockdown of At4g37990 gene for confirming radiation exposure, prepared by transforming an RNAi (RNA interference) expression vector containing an At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene into Arabidopsis thaliana plants. To provide a transgenic plant.

Another object of the present invention,

1) identifying a function of increasing expression under irradiation conditions of the At4g37990 gene;

2) exposing the transgenic plant of the invention to a region of suspected radiation exposure;

3) analyzing the At4g37990 gene expression of the transgenic plant of step 2); And

4) to provide a method for confirming radiation exposure comprising the step of comparing the expression level of the gene of step 3) with the expression level of the At4g37990 gene of a control plant not exposed to the area of step 2).

Still another object of the present invention is a kit for confirming radiation exposure including an At4g37990 gene or a complementary strand molecule thereof, and a primer or probe specifically binding to the At4g37990 gene, or an At4g37990 gene encoded by the gene. It is to provide a kit for confirming radiation exposure including an antibody (antibody) or aptamer (aptamer) that specifically binds to a protein.

Still another object of the present invention is to measure the At4g37990 gene expression level of a group of plants exposed to radiation by using a kit including a primer for the At4g37990 gene sequence conserved region. It is to provide a method for checking radiation exposure.

In order to achieve the above object, the present invention is prepared by transforming RNAi (RNA interference) expression vector containing At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene to Arabidopsis plants, At4g37990 gene knockdown ( knockdown) transgenic plants are provided.

In addition,

1) identifying a function of increasing expression under irradiation conditions of the At4g37990 gene;

2) exposing the transgenic plant of the invention to a region of suspected radiation exposure;

3) analyzing the At4g37990 gene expression of the transgenic plant of step 2); And

4) provides a method for confirming radiation exposure comprising comparing the expression level of the gene of step 3) with the At4g37990 gene of the control plant not exposed to the area of step 2).

In addition, the present invention provides a kit for confirming radiation exposure comprising the At4g37990 gene or its complementary strand molecule.

In addition, the present invention includes a primer or probe specifically binding to the At4g37990 gene, or an antibody or aptamer specifically binding to a protein encoded by the At4g37990 gene. Provide kits for radiation exposure checks.

In addition, the present invention using a kit comprising a primer for the At4g37990 gene sequence common region (conserved region), whether the radiation exposure of the plant group comprising the step of measuring the At4g37990 gene expression level of the plant group exposed to radiation Provide a verification method.

By confirming that the expression of the At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene according to the present invention is increased in the irradiation conditions, by identifying the function as a radiolabeled gene of the gene, to confirm the radiation exposure using the radiolabeled gene Therefore, it can be seen that by producing a transgenic plant using the radiolabeled gene it can be usefully used as an indicator plant for detecting the exposure of radiation in the plant ecosystem.

1 is a diagram showing the results of verifying the expression pattern according to the irradiation dose of the At4g37990 gene by reverse transcriptase (RT-PCR):
Values in () are the analysis results of the microarray expression (fold change) of the gene between the control plant (non-gamma irradiated) and the gamma irradiated plant.
Figure 2 is a diagram showing the principle of the BP response and LR response of the transform vector production process for RNAi (RNA interference) technology.
Figure 3 is a diagram showing a detailed schematic of the construction of the At4g37990 gene knockdown vector of the present invention.
Figure 4 is a diagram showing the final line selection process through the expression changes of the At4g37990 gene knockdown T3 transgenic plant under gamma irradiation and non-irradiation conditions.
Figure 5 is a diagram showing the growth difference compared to the control after 24 days germination of the At4g37990 gene knockdown transgenic plants (left: control and right: transgenic plants of the present invention).
Figure 6 is a diagram showing the growth difference compared to the control after 35 days germination of At4g37990 gene knockdown transgenic plants (left: control and right: transgenic plants of the present invention).
7 is a reverse transcriptase polymerase reaction (RT-) in which the radiation levels of 10, 25, 100, 200, 800, 2000, and 4000 Gy doses of the At4g37990 gene knockdown transgenic plants were re-examined to identify the expression of the genes. PCR) results are shown.

Hereinafter, the present invention will be described in detail.

The present invention provides an At4g37990 gene knockdown transformed plant for confirmation of radiation exposure, which is prepared by transforming an RNAi (RNA interference) expression vector comprising an At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene into a Arabidopsis plant. do.

The At4g37990 gene preferably has a nucleotide sequence set forth in SEQ ID NO: 1, but is not limited thereto.

The At4g37990 gene is Arabidopsis thaliana ), but is not limited thereto.

The transformation may be a conventional plant transformation method known to those skilled in the art, such as Agrobacterium mediated method, gene gun PEG method and electroporation (electroporation) can be used. (Horsch, et al., Cold Spring Harb Symp Quant Biol., 50: 433-7, 1985; Rogerset, et. Al., 1986), but introducing the vector into Agrobacterium; And co-culturing the Agrobacterium with a plant or culture cells (including callus, callus), thereby preparing a transformed plant.

Such vectors are used to refer to DNA fragment (s), nucleic acid molecules that are delivered into cells. The vector replicates DNA and can be reproduced independently in a host cell and usually has the same meaning as a carrier.

An expression vector also refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium, can transfer some of their so-called T-region to plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the At4g37990 gene according to the invention into a plant host are viral vectors such as those derived from double stranded plant viruses (e.g., CaMV) and single stranded viruses, gemini viruses, etc., For example, it may be selected from incomplete plant viral vectors, the use of which may be used, but is not limited to, particularly when it is difficult to properly transform the plant host.

In addition, the vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, such as all genes that can distinguish the transformed cells from non-transformed cells, for example, glyphosate (Glyphosate) Or antibiotic resistance genes such as herbicide resistance genes such as Phosphinotricin, kanamycin, G418, Bleomycin, Hygromycin, Chloramphenicol, etc. .

The transgenic plant may be obtained through a sexual breeding method or an asexual breeding method which is a conventional method in the art. More specifically, the plant of the present invention can be obtained through the oily breeding process of producing seeds through the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant vector comprising the At4g37990 gene according to the present invention can be obtained through the asexual propagation method which is a process of induction, rooting and soil purification of the callus according to a conventional method. That is, the fragments of the plant transformed with the recombinant vector containing the At4g37990 gene are placed in a suitable medium known in the art, and then cultured under appropriate conditions to induce the formation of callus, and when the shoots are formed, they are transferred to a hormone-free medium. Incubate. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. Plants with accelerated development or differentiation can be obtained by inducing roots and then transplanting them into the soil to purify them. In the present invention, the transgenic plant preferably includes, but is not limited to, a whole plant as well as tissues, cells or seeds that can be obtained therefrom.

In a specific embodiment of the present invention, in order to test the growth reaction under the irradiation conditions of the trophic Arabidopsis, gamma rays were irradiated at various doses, and growth was promoted under 100 Gy irradiation conditions, and growth was inhibited under 800 Gy irradiation conditions. It confirmed that it became.

In another specific embodiment of the present invention, in order to search for gamma-ray-specific genes, after irradiating gamma rays to the genital tract Arabidopsis plants, RNA was extracted and microarray analysis was performed to find common results at 100 Gy and 800 Gy. Gamma-ray specific At4g37990 gene with increased expression was selected.

In another specific embodiment of the present invention, the reverse transcriptase polymerase chain reaction (RT-PCR) was performed to confirm the expression pattern after gamma irradiation of the At4g37990 gene. It was confirmed that the increase by (see Figure 1).

In another specific embodiment of the present invention, an RNAi expression vector for knocking down the At4g37990 gene was prepared using an Invitrogen Gateway cloning technology method to prepare an RNAi expression vector into which the At4g37990 gene was introduced (FIG. 2 and 3).

In another specific embodiment of the present invention, in order to prepare the At4g37990 gene knockdown transformed plant, the transgenic plant is transformed into an RNAi expression vector for At4g37990 gene knockdown of the present invention through a transformation method using Agrobacterium (GV3101). Was made (see FIG. 4).

In another specific embodiment of the present invention, in order to analyze the growth of the At4g37990 gene knockdown transformed plant of the present invention, after germinating the seed of the transformed plant of the present invention, the growth difference is compared with the control plant, It was confirmed that the conversion plants progressed to the reproductive stage somewhat faster than the control plants (see FIG. 5), and 35 days after germination, it was confirmed that there was more difference in plant size (see FIG. 6). However, in the case of transgenic plants, the growth was faster than that of the control plants, and although the fire was normally generated, the development of the silique was delayed (see FIG. 6 (right picture)).

In another specific embodiment of the present invention, in order to reexamine whether the At4g37990 gene knockdown transgenic plant is expressed by radiation, the nutrient growth stage of the knockdown transgenic plant of the present invention is 10, 25, 100, 200, 800, The reverse transcriptase polymerase reaction (RT-PCR), which revealed the increased expression of the gene by re-irradiating 2000 and 4000 Gy doses, revealed that the gene was expressed in knockdown transgenic plants under various irradiation conditions compared to the control plants. This significantly increased (see FIG. 7).

Therefore, the At4g37990 gene knockdown transformed plant prepared by transforming an RNAi (RNA interference) expression vector containing the At4g37990 gene of the present invention into Arabidopsis plants is effectively knocked down in the expression of the At4g37990 gene under non-irradiated conditions. Since the expression is increased in the gamma-irradiation conditions, it can be usefully used as an indicator plant to determine whether gamma-ray exposure in the ecosystem.

In addition,

1) identifying a function of increasing expression under irradiation conditions of the At4g37990 gene;

2) exposing the transgenic plant of the invention to a region of suspected radiation exposure;

3) analyzing the At4g37990 gene expression of the transgenic plant of step 2); And

4) provides a method for confirming radiation exposure comprising comparing the expression level of the gene of step 3) with the At4g37990 gene of the control plant not exposed to the area of step 2).

At4g37990 gene of the transgenic plant of the present invention increases the gamma ray-specific expression, so the transgenic plants in areas suspected to be exposed to radiation and At4g37990 gene expression analysis of plants in areas not exposed to radiation to confirm radiation exposure It can be usefully used.

In addition, the present invention provides a kit for confirming radiation exposure including At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene or its complementary strand molecule.

In addition, the present invention includes a primer or probe specifically binding to the At4g37990 gene, or an antibody or aptamer specifically binding to a protein encoded by the At4g37990 gene. Provide kits for radiation exposure checks.

It is preferred to have the nucleotide sequence described in At4g37990 gene SEQ ID NO: 1, but is not limited thereto.

The At4g37990 gene is preferably derived from Arabidopsis but not limited thereto.

The At4g37990 gene is preferably increased in response to radiation exposure, but is not limited thereto.

The radiation is preferably gamma rays, electron beams, UV or X-rays, more preferably gamma rays, but is not limited thereto.

The radiation exposure checking kit may further include one or more substances and reagents for detecting a reaction product and instructions for reacting with the At4g37990 gene, and one or more substances that react with the At4g37990 gene may be RNA or DNA of the At4g37990 gene. RNA or DNA complementary to, and can be specifically bound to a protein encoded by the At4g37990 gene and the reaction product detection reagent may be a nucleic acid or protein labeling and coloring reagent, but is not limited thereto.

The primer is a nucleic acid sequence having a short free 3-hydroxyl group, which forms a complementary template and base pair and serves as a starting point for template strand copying. DNA synthesis can be initiated in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at the appropriate buffer and temperature. Therefore, PCR amplification can be performed using the sense and antisense primers of the polynucleotide of the At4g37990 gene of the present invention to confirm radiation exposure through the generation of a desired product, and the length of PCR conditions, sense and antisense primers is Modifications can be made as appropriate based on what is known in the specification, but is not limited thereto.

The probe refers to a nucleic acid fragment such as RNA or DNA, which is short to several bases to hundreds of bases, which can achieve specific binding with mRNA, and is labeled to indicate the presence or absence of a specific mRNA. You can check. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc., but are not limited thereto. Therefore, hybridization may be performed by using a probe complementary to the polynucleotide of the At4g37990 gene of the present invention to determine whether the radiation is exposed through hybridization, and selection of a suitable probe and hybridization conditions are based on those known in the art. Modifications may be made but are not limited thereto.

The primer or probe may be chemically synthesized using, but not limited to, phosphoramidite solid support methods, or other well known methods. In addition, such nucleic acid sequences can also be modified using many means known in the art. Such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphotriester, phosphoramidate, carbamate Etc.) or modifications with charged linkers (eg, phosphorothioate, phosphorodithioate, etc.) are preferred, but not limited thereto.

Preferably, the primer or probe comprises 8 or more nucleotides, and the hybridization method can be achieved by exposing or contacting the primer or probe to a polynucleotide of the At4g37990 gene of the present invention. Preferably these sequences are hybridized under appropriately controlled conditions to minimize nonspecific binding, and suitable conditions for detection of, for example, about 80% to 90% of the same sequence are 0.25 M Na ₂ HPO ₄ , pH 7.2, 6.5 Overnight hybridization at 42 ° C. in% SDS, 10% dextran sulfate and final wash at 55 ° C. in 0.1 × SSC, 0.1% SDS. In addition, suitable conditions for detection of at least about 90% of the same sequence are hybridized overnight at 65 ° C. in 0.25 M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate, and 0.1 × SSC, 0.1% SDS It may include, but not limited to, the final washing at 60 ℃.

In a specific embodiment of the present invention, in order to test the growth reaction under the irradiation conditions of the trophic Arabidopsis, gamma rays were irradiated at various doses, and growth was promoted under 100 Gy irradiation conditions, and growth was inhibited under 800 Gy irradiation conditions. It confirmed that it became.

In another specific embodiment of the present invention, in order to search for gamma ray-specific genes, after irradiating gamma rays to Arabidopsis plants, RNA was extracted and microarray analysis was performed. As a result, expression levels were commonly expressed at 100 Gy and 800 Gy. Gamma-ray specific At4g37990 gene with high (fold change) was selected.

In another specific embodiment of the present invention, the reverse transcriptase polymerase chain reaction (RT-PCR) was performed to confirm the expression pattern after gamma irradiation of the At4g37990 gene. It was confirmed that the increase by (see Figure 1).

Therefore, the At4g37990 gene of the present invention can be usefully used for a kit for confirming radiation exposure because gamma-ray-specific expression is increased.

In addition, the present invention using a kit comprising a primer for the At4g37990 gene sequence common region (conserved region), whether the radiation exposure of the plant group comprising the step of measuring the At4g37990 gene expression level of the plant group exposed to radiation Provide a verification method.

Measuring the expression level is to determine the expression level of the At4g37990 gene in the test plants to confirm the radiation exposure, it is preferable to measure by gene amplification or antigen-antibody reaction, RT-PCR, competitive RT-PCR ( Competitive RT-PCR, Quantitative real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, Western blotting , ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation assay), complement fixation assay (Complement Fixation Assay), FACS, protein chip (protein chip) is more preferred, but is not limited thereto.

In a specific embodiment of the present invention, in order to test the growth reaction under the irradiation conditions of the trophic Arabidopsis, gamma rays were irradiated at various doses, and growth was promoted under 100 Gy irradiation conditions, and growth was inhibited under 800 Gy irradiation conditions. It confirmed that it became.

In another specific embodiment of the present invention, in order to search for gamma ray-specific genes, after irradiating gamma rays to Arabidopsis plants, RNA was extracted and microarray analysis was performed. As a result, the expression was commonly expressed at 100 Gy and 800 Gy. Increasing gamma-ray specific At4g37990 gene was selected.

In another specific embodiment of the present invention, the reverse transcriptase polymerase chain reaction (RT-PCR) was performed to confirm the expression pattern after gamma irradiation of the At4g37990 gene. It was confirmed that the increase by (see Figure 1).

Therefore, the expression of the At4g37990 gene of the present invention increases gamma ray-specific expression, and after measuring the At4g37990 gene expression level from a test plant suspected of being exposed to radiation, compared with the expression level of the At4g37990 gene of a control group not exposed to radiation. It can be useful for checking radiation exposure.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

< Example  1> Radiation and Growth Reaction Test

Arabidopsis , a model plant thaliana ) (Provided by Kumho Research Institute of Chonnam National University, Lansdberg erecta ecotype) in the plant tissue culture room under 22 ° C (18 hours: light condition, 6 hours: dark condition) growth conditions, sowing 7 seeds per 15 cm diameter in pot, so that gamma rays (100, 200, 300, 400, 800, 1200, 1600 and 2000 Gy). The gamma-irradiation facility used a low-level gamma-ray irradiation apparatus device of the Korea Atomic Energy Research Institute, and the ⁶⁰ Co was used as the gamma-ray source.

As a result, it was confirmed that growth was rather promoted under 100 Gy irradiation conditions, and growth was completely suppressed at a dose of 800 Gy or more. Therefore, in order to mass-assay genes that specifically react to gamma rays, 100 Gy and 800 Gy were determined as appropriate doses, and the following experiment was conducted.

< Example  2> Screening Gamma Specific Reaction Gene

In order to select gamma-specific genes, microarray analysis was performed.

Specifically, gamma rays of 100 Gy and 800 Gy were irradiated to the Arabidopsis plants of the nutrient growth period in the same manner as in <Example 1>. Then, 2 days after gamma irradiation, RNA was extracted from plant tissues in the ground and microarray analysis was performed for mass assay of genes that specifically react with gamma rays. The chip for microarray analysis was commercialized using Agilent oligochip (22k), and after hybridization, image quantification was performed using GenePix Pro 6.0 software, and each spot using LOWESS program. Normalization of was performed. In addition, the microarray analysis was performed twice.

As a result, the At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene, which is described as the nucleotide sequence of SEQ ID NO. 1 having a high fold change, was selected among the genes with increased expression in gamma irradiation at 100 Gy and 800 Gy, respectively. .

< Example  3> according to gamma irradiation dose At4g37990  Confirmation of gene expression

In order to confirm the expression pattern according to gamma irradiation dose of the At4g37990 gene selected in <Example 2>, reverse transcriptase polymerase chain reaction (RT-PCR) was performed after gamma irradiation of 100 Gy and 800 Gy, respectively.

Specifically, the whole plant RNA was extracted using TRI reagent, and the actual expression after γ-irradiation of At4g37990 gene was quantitatively compared and examined through reverse transcriptase polymerase chain reaction. Reverse transcriptase polymerase chain reaction was carried out according to the Access RT-PCR system (promega) with 0.5 ㎍ of extracted RNA as a template, and cDNA synthesis and polymerase chain reaction amplification were carried out in a tube with a specific primer (SEQ ID NO: 2 and SEQ ID NO: 3), and the total volume of the reaction was performed at 25 μl. In addition, PCR cDNA synthesis was -45 ℃ 50 minutes / pre-denaturation-94 ℃, 2 minutes / 30 cycles (denaturation-94 ℃, 30 seconds / annealing-55 ℃, 30 seconds / extension-72 ℃, 1 minute), Final extension was carried out under conditions such as 72 ° C. and 7 minutes.

At4g37990 forward primer: 5'-GAAGATCGTGACATTGGTACTCGTCG-3 '(SEQ ID NO: 2); And

At4g37990 reverse primer: 5'-CAACAAGTGGGATAAGCAACATTGC-3 '(SEQ ID NO: 3).

As a result, as shown in Figure 1, the At4g37990 gene was confirmed that the expression of the gene is significantly increased after gamma irradiation compared to the control (non-gamma irradiated) (Fig. 1).

< Example  4> At4g37990  Gene knockdown ( knockdown )for someone RNAi  Preparation of Plant Transgenic Carriers

In order to prepare an RNAi plant transformation carrier for knockdown of At4g37990 gene, the following cloning method of the gateway cloning technology (Invitrogen), which is a method of cloning using a process in which phage DNA is integrated into the bacterial genome, is described. Was used.

Specifically, the genes contained between the att sequences are exchanged by cross-reaction between genes having a specific sequence in vitro by the action of Recombinase, and the BP and LR reactions are used. The principle is as shown in FIG. The LR reaction means a cross reaction between attL and attR, and the BP reaction means a cross reaction between attB and attP. On this principle, the desired gene can be inserted into the carrier to finally knock down the gene. Thus, the Arabidopsis cDNA was templated and PCR amplified using the following primers (SEQ ID NO: 4 and SEQ ID NO: 5) (underlined nucleotide sequence means att sequence and then the gene was At4g37990 gene specific nucleotide sequence). to be).

At4g37990_BP_Forward primer:

5'- GGGGACAAGTTTGTACAAAAAAGCAGGCT TTTGAGAGGAAGATGGTAATGGG-3 '(SEQ ID NO: 4); And

At4g37990_BP_Reverse primer:

5′- GGGGACCACTTTGTACAAGAAAGCTGGGT ACATTTTTGGTATTTAACAGTAC-3 ′ (SEQ ID NO: 5).

The PCR product was cloned into pDONR207 Donor carrier (Invitrogen) to induce the BP reaction to prepare the entry clone into the plasmid (preparation) and confirm the accuracy of the sequence. Then, the LR reaction between the entry clone and the pNW2300 / ANDA Destination carrier was induced to confirm the cloning of the gene and the accuracy of the sequence.

As a result, as shown in Fig. 3, a carrier for knocking down the At4g37990 gene was prepared (Fig. 3).

< Example  5> At4g37990  Gene knockdown transgenic plant production and selection

In order to manufacture the At4g37990 gene knockdown transformed plants, Arabidopsis transformants were prepared by using the Agrobacterium (GV3101), floral dip transformation method, and the kanamycin. Resistant selection allows the selection of homoline to finally obtain six T3 pure compounds (# 19-11, # 20-11, # 34-5, # 42-5, # 47-3, # 60-5) It was. Then, reverse transcriptase polymerase chain reaction [PCR cDNA synthesis -45 ℃ 50 minutes / pre-denaturation-94 ℃, 2 minutes / 30 cycles (denaturation-94 ℃, 30 seconds / annealing-55 ℃, 30 seconds / extension-72 ℃, 1 min), final extension-72 ℃, 7 minutes] to express the expression of At4g37990 knock-down expression in the non-irradiated conditions compared to the control plants and to increase the expression of the gene under 800 Gy irradiation conditions I wanted to select.

As a result, as shown in FIG. 4, the expression of At4g37990 was knocked down compared to the control plants in the non-irradiated conditions, and the expression of the gene was significantly increased in comparison with other strains at 800 Gy gamma-irradiated conditions. # 34-5 line transgenic plants were finally selected (FIG. 4).

< Example  6> knockdown ( Knockdown Transgenic Plant Growth Analysis

In order to test the characteristics of the # 34-5 transgenic plant finally selected in <Example 5> in the general growing condition, the growth difference was assayed 24 and 35 days after germination of seeds.

As a result, as shown in FIG. 5 and FIG. 6, in the case of 24 days after germination, it was confirmed that the transgenic plants progressed to the reproductive stage somewhat faster than the control plants (FIG. 5). It was confirmed that there are more differences in plant size (Fig. 6). However, in the case of transgenic plants, the growth was faster than that of the control plants, and although the fire was normally generated, the development of the silique was delayed (FIG. 6 (right picture)).

< Example  7> following radiation re-investigation At4g37990  Gene expression test

In order to re-investigate the responsiveness to radiation in nutrient growing plants by sowing seeds of the T34 homo lineage of the # 34-5 lineage, which was finally determined to be effectively knocked down, the At4g37990 gene selected in Example 5 above. RNA was extracted from trophic Arabidopsis plants that were irradiated with 100, 200, 800, 2000, and 4000 Gy of radiation, respectively, and subjected to reverse transcription polymerase chain reaction [PCR cDNA synthesis -45 ° C 50 min / pre-denaturation-94 ℃, 2 minutes / 30 cycles (denaturation-94 ℃, 30 seconds / annealing-55 ℃, 30 seconds / extension-72 ℃, 1 minute), final extension-72 ℃, 7 minutes] to perform the final expression of At4g37990 Verified by.

As a result, as shown in Figure 7, the knockdown transgenic plants were observed to increase the expression of the gene of interest under various irradiation conditions compared to the control plants (Fig. 7). It was found that the expression of the gene is increased in the irradiation conditions even in the case of knockdown plants including general plants.

<110> Korea atomic energy research institute <120> Investigation of Radio marker gene At4g37990-aromatic          alcohol: NADP + oxidoreductase as a Radiomarker gene and transgenic          indicator plant using the gene <130> 11p-05-23 <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 1080 <212> DNA <213> Arabidopsis thaliana <400> 1 atgggaaagg ttcttcagaa agaggcgttc ggattagccg cgaaagacaa ttccggagtt 60 ctctcgcctt tcagtttcac tagaagggaa acaggagaaa aggatgtgag gttcaaagtg 120 ttattttgtg gaatttgtca ctctgatttg catatggtca agaacgagtg gggaatgtct 180 acttatcctc ttgtcccagg gcatgagatc gtgggcgtgg tgactgaagt cggagccaaa 240 gtgactaaat tcaaaaccgg agaaaaagtc ggagttgggt gtttggtcag ctcgtgcggg 300 tcatgtgaca gctgcaccga aggtatggaa aactattgtc caaaatcaat ccaaacgtac 360 ggattcccgt actacgacaa caccataaca tacggtggtt actccgacca catggtttgc 420 gaggaaggtt tcgtcatccg tattccagac aatctcccct tggacgccgc cgcaccgctc 480 ctctgtgccg gtatcacggt ctattcccct atgaagtatc acgggctcga caaacccggt 540 atgcacatcg gtgtggtagg attaggcggt ttaggtcatg taggagtgaa atttgccaag 600 gctatgggta ctaaggttac ggttattagt acttcggaga aaaagagaga tgaggcgatt 660 aatcggcttg gtgcggatgc tttcttggtg agccgtgacc caaaacagat taaggatgca 720 atgggtacta tggatggtat aattgatacc gtctctgcga ctcattcact tcttccgttg 780 ctcggtttgc tgaagcataa gggaaaactt gttatggttg gtgcacccga gaagccactc 840 gagctacctg tcatgcctct catctttgag aggaagatgg taatgggaag tatgatagga 900 gggataaaag agacccagga aatgatagat atggccggga aacacaacat cactgcggat 960 attgagctta tctctgccga ttatgtcaac accgccatgg aacggctaga gaaagccgac 1020 gttaggtacc gctttgtgat tgatgttgcc aacacattga agcctaatcc taatttataa 1080                                                                         1080 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> At4g37990 forward primer <400> 2 gaagatcgtg acattggtac tcgtcg 26 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> At4g37990 reverse primer <400> 3 caacaagtgg gataagcaac attgc 25 <210> 4 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> At4g37990 BP forward primer <400> 4 ggggacaagt ttgtacaaaa aagcaggctt ttgagaggaa gatggtaatg gg 52 <210> 5 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> At4g37990 BP reverse primer <400> 5 ggggaccact ttgtacaaga aagctgggta catttttggt atttaacagt ac 52

Claims (14)

An RNAi (RNA interference) expression vector containing the At4g37990 (aromatic alcohol: NADP + oxidoreductase) gene was transferred to Arabidopsis. thaliana ) At4g37990 gene knockdown transgenic plant for confirmation of radiation exposure, prepared by transforming the plant.
The At4g37990 gene knockdown transformed plant according to claim 1, wherein the At4g37990 gene has a nucleotide sequence as set forth in SEQ ID NO: 1.
2. The At4g37990 gene knockdown transformed plant according to claim 1, wherein the transformation uses an Agrobacterium mediated transformation technique.
1) identifying a function of increasing expression under irradiation conditions of the At4g37990 gene;
2) exposing the transgenic plant of claim 1 to an area of suspected radiation exposure;
3) analyzing the At4g37990 gene expression of the transgenic plant of step 1); And
4) comparing the expression of the gene of step 3) with the At4g37990 gene of the control plant that is not exposed to the region of step 2) the expression of radiation exposure comprising the step of comparing.
The method of claim 4, wherein the gene expression analysis is performed through gene amplification or antigen-antibody reaction.
Kit for confirming radiation exposure comprising the At4g37990 gene or its complementary strand molecule.
For radiation exposure including a primer or probe specifically binding to the At4g37990 gene, or an antibody or aptamer specifically binding to a protein encoded by the At4g37990 gene Kit.
The kit for confirming radiation exposure according to claim 6 or 7, wherein the gene has a nucleotide sequence as set forth in SEQ ID NO: 1.
8. The kit according to claim 6, wherein the At4g37990 gene is derived from a Arabidopsis vulgaris.
The kit for confirming radiation exposure according to claim 6 or 7, wherein the At4g37990 gene has an increased expression level when exposed to radiation.
8. The kit according to claim 6 or 7, wherein the radiation is any one selected from the group consisting of gamma rays, electron beams, UV rays and X-rays.
The kit according to claim 11, wherein the radiation is gamma rays.
Using a kit comprising a primer for the At4g37990 gene sequence common region (conserved region), a method for checking the radiation exposure of the plant group comprising the step of measuring the expression level of the At4g37990 gene of the plant group exposed to radiation.
The method of claim 13, wherein the expression level is measured by gene amplification or antigen-antibody reaction.

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