KR101276733B1 - 17β-estradiol(E2) responsive genes in Oryzias javanicus and the method for diagnosing environment pollution using the same - Google Patents

Abstract

The present invention relates to genes of sea pods corresponding to exposure of 17β-estradiol (E2) and methods for diagnosing environmental pollution using the same. Specifically, biomarkers corresponding to marine environmental pollution in sea pods To find and detect specific gene candidates for 17β-estradiol exposure, HazChem Fish Array was used to identify biomarkers and changes in expression levels of these gene biomarkers. Since it has been identified, it can be used for diagnosis of environmental pollution.

Description

{17β-estradiol (E2) responsive genes in Oryzias javanicus and the method for diagnosing environment pollution using the same} of seafish genes corresponding to exposure to 17β-estradiol (E2)

The present invention is to confirm whether the exposure to 17β- estradiol (E2), a kind of endocrine disrupting chemical (EDC), environmental pollution detection or diagnostic microarray chip and exposure detection and environment using the same The present invention relates to a contamination diagnosis method.

17β-estradiol (E2) is the most potent estrogen class of steroid hormones (estrone, E1, 17β-estradiol and estriol, E3) in mammals. In humans, it is mainly formed in the ovaries and placenta of women of childbearing age, but also in the adipose tissue of men and menopausal women. Synthesized form of 17β-estradiol has been used to control various symptoms associated with menopause in women, such as vascular dyskinesia, vulva and vaginal contraction disorders, osteoporosis and the like. Estrone and estriol including 17β-estradiol secreted by humans and animals are discharged to the environment by sewage treatment plants and animal wastes. These released steroid hormones pose a threat to the stability of the ecosystem. In other words, the normal endocrine system function of males is inhibited through the action such as vitellogenin synthesis and induction of femaleization in male fish, which results in the reproductive ability of males being lowered and disturbing the ecosystem. It is expected. Therefore, there is a need to develop a method for predicting the presence of 17β-estradiol, predicting the biological change caused by exposure, and early diagnosis.

Sea pods (or Oryzias javanicus ) are fish that can inhabit all of the world's water systems (freshwater and seawater) (Inoue and Takei, Zoological). Sci . , 19, 727-734, 2002) and is expected to be applicable to environmental risk assessment of toxic chemicals in freshwater and seawater. To date, studies on changes in fertility and changes in vitelogenin protein concentrations in male liver tissues for exposure to 17β-estradiol (Imai et al . , Mar. Poll. Bull ., 51, 708-714, 2005); 17β- estradiol nose Rio angiogenin (choriogenin) Expression of H and L gene in the tissue between the male by exposure diol (like Yu, Aquat. Toxicol., 77 , 348-358, 2006), liver tissue for a variety of heavy metal exposure Expression of Metallothionein Genes in Humans (Woo et al . , Mar. Biotech . , 8, 654-662, 2006) and Vitellogenin protein in liver fertility and liver tissues by exposure to estrone in the environment A study of concentration changes (Imai et al . , Environ . Toxicol . Chem ., 26, 726-731, 2007) has been reported. Also, after exposure to 6 major heavy metals (Ag, Cd, Cr, Cu, Ni, Zn) (Woo et al., Comp. Biochem. Physiol. C, 149, 289-299, 2009) (Iprobenfos) exposure (Woo et al., Comp. Biochem. Physiol. C, 149, 427-432, 2009) and toxaphene exposure to polychlorinated camphor (Woo et al., Comp. Biochem. Physiol. C, 153, 355 -361, 2011), there are reports of changes in the expression levels of antioxidant enzymes and stress-related genes.

Biologists have evolved biological defense mechanisms to maintain homeostasis in response to changes in the external environment, such as environmental pollution, climate change, and infection with pathogenic microorganisms. Most of these mechanisms of defense appear to modulate the amount of a specific protein by actively changing the expression level of a particular gene. Therefore, by identifying specific genes for specific changes and monitoring changes in their expression levels, you can get not only information about environmental changes in specific regions, but also information on the impact of these environmental changes on life phenomena and ecosystem health. Can be. To date, no research has been conducted on the acquisition and detection of specific gene candidates for exposure to 17β-estradiol in sea pods.

Accordingly, the present inventors attempted to discover biomarkers corresponding to marine environmental pollution in sea pods, specifically, 17β using HazChem Fish Array to secure and detect specific gene candidates for 17β-estradiol exposure. The present invention was completed by confirming changes in expression levels of biomarkers and gene biomarkers capable of confirming exposure to estradiol, and confirming that they can be used for stress detection and diagnosis of environmental pollution.

An object of the present invention is to provide a gene of Oryzias javanicus corresponding to 17β-estradiol (E2) exposure and stress detection and environmental pollution diagnostic method using the same.

In order to achieve the above object, the present invention provides an oligonucleotide comprising all or a portion of a gene sequence whose expression is increased or decreased in response to estrogen-based steroid hormone exposure, or oligonucleotide molecules complementary to the oligonucleotide The present invention provides a microarray chip for detecting stress and diagnosing environmental pollution caused by exposure to steroid hormones of the estrogen family.

The present invention also provides a method for detecting stress and diagnosing environmental pollution according to steroid hormone exposure of an estrogen family using the gene.

In addition, the present invention, the micro-array chip, estrogen-based

The present invention provides a kit for detecting stress and diagnosing environmental pollution due to steroid hormone exposure.

In addition, the present invention provides a kit for stress detection and environmental pollution diagnosis according to steroid hormone exposure of the estrogen-based, comprising a primer pair that is complementary to the gene and can amplify the gene.

We attempted to discover biomarkers in response to marine environmental pollution in sea pods. Specifically, we used HazChem Fish Array to secure and detect specific gene candidates for 17β-estradiol (E2) exposure. Therefore, since biomarkers capable of confirming exposure to 17β-estradiol and changes in expression levels of these gene biomarkers were identified, they can be used in a method for diagnosing environmental pollution.

Hereinafter, the present invention will be described in detail.

The present invention provides a gene for detecting the exposure of steroid hormones of the estrogen series in the sample and diagnosing environmental pollution, consisting of the following groups:

The gene described by SEQ ID NO: 1 (Dimethylglycine dehydrogenase), the gene described by SEQ ID NO: 2 (Fructose-bisphosphate aldolase B), the gene described by SEQ ID NO: 3 (Fatty acid binding protein 10 liver basic), described by SEQ ID NO: 4 Gene (Claudin), gene described by SEQ ID NO: 5 (Cytochrome P450 2P3), gene described by SEQ ID NO: 6 (Aldolase B), gene described by SEQ ID NO: 7 (Cytochrome c-1, cyc1), SEQ ID NO: 8 The gene described (Selenoprotein M), the gene described in SEQ ID NO: 9 (ATPase H + transporting V1 subunit F, atp6v1f), the gene described in SEQ ID NO: 10 (Cytochrome oxidase subunit I, COI), the gene described in SEQ ID NO: 11 ATP citrate lyase isoform 2), the gene described by SEQ ID NO: 12 (Ribosomal protein L13a, rpl13a), the gene described by SEQ ID NO: 13 (Cytochrome c oxidase subunit I), the gene described by SEQ ID NO: 14 (Pyrroline-5-carboxylate r eductase 1, pycr1), the gene described by SEQ ID NO: 15 (Exs-related protein), the gene described by SEQ ID NO: 16 (Cysteine-rich with EGF-like domains 2, creld2), the gene described by SEQ ID NO: 17 (Selenoprotein 15), the gene described by SEQ ID NO: 18 (Beta-galactoside-binding lectin), the gene described by SEQ ID NO: 19 (hv1 gene for histone H2 variant), the gene described by SEQ ID NO: 20 (LAG1 longevity assurance 2), sequence The gene described in SEQ ID NO: 21 (Inositol oxygenase), the gene described in SEQ ID NO: 22 (Acyl-CoA synthetase long-chain family member 1), the gene described in SEQ ID NO: 23 (Tetraspanin-3), described in SEQ ID NO: 24 Microsomal triglyceride transfer protein, gene described by SEQ ID NO: 25 (Amino-terminal enhancer of split), gene described by SEQ ID NO: 26 (non-classical MHC class I antigen), gene described by SEQ ID NO: 27 (NADH -cytochrome b5 reductase 3), standing The gene described in SEQ ID NO: 28 (rRNA 2-O-methyltransferase fibrillarin), the gene described in SEQ ID NO: 29 (Vitellogenin II, vit-6), the gene described in SEQ ID NO: 30 (Choriogenin H minor), described in SEQ ID NO: 31 Gene (Adenylosuccinate synthetase isozyme 2), gene described by SEQ ID NO: 32 (Mannose-P-dolichol utilization defect 1b, mpdu1b), gene described by SEQ ID NO: 33 (RAB3 GTPase activating protein subunit 2 (non-catalytic)), Gene described by SEQ ID NO: 34 (Tax1 (human T-cell leukemia virus type I) binding protein 1b), Gene described by SEQ ID NO: 35 (40S ribosomal protein S24-like protein), Gene described by SEQ ID NO: 36 (Exosome complex exonuclease RRP4), gene described by SEQ ID NO: 37 (Iduronate 2-sulfatase precursor), gene described by SEQ ID NO: 38 (Vitellogenin 1, ol-vit1), gene described by SEQ ID NO: 39 (Oligosaccharyltransferase complex subunit, ostc) , Gene described by SEQ ID NO: 40 (Abhydrolase domain containing 11), Gene described by SEQ ID NO: 41 (B-cell antigen receptor complex-associated protein alpha-chain precursor), Gene described by SEQ ID NO: 42 (Alpha-2,3 -sialyltransferase ST3Gal I), the gene described in SEQ ID NO: 43 (Glutathione reductase (mitochondrial)), the gene described in SEQ ID NO: 44 (Choriogenin L), the gene described in SEQ ID NO: 45 (Integral membrane protein 1), SEQ ID NO: 46 (Complement control protein factor I-B), gene described in SEQ ID NO: 47 (Glutamate receptor, ionotropic, delta 1, GRID1), gene described in SEQ ID NO: 48 (Dolichyl-alpha-1,6-mannosyltransferase (alg12)), the gene described by SEQ ID NO: 49 (Macrosialin precursor), the gene described by SEQ ID NO: 50 (Metalloreductase STEAP4), the gene described by SEQ ID NO: 51 (14-alpha demethylase (CYP51)), and SEQ ID NO: 52 Listed Gene (Bromodomain containing 2 (RING3)), gene described by SEQ ID NO: 53 (Apolipoprotein B), gene described by SEQ ID NO: 54 (Glutamate dehydrogenase 1b), gene described by SEQ ID NO: 55 (Glucose-6-phosphate dehydrogenase) And the gene set forth in SEQ ID NO: 56 (Transferrin).

In the gene group, a gene whose expression is increased in response to an estrogen-based steroid hormone may include, but is not limited to:

The gene described in SEQ ID NO: 7 (Cytochrome c-1, cyc1), the gene described in SEQ ID NO: 8 (Selenoprotein M), the gene described in SEQ ID NO: 9 (ATPase H + transporting V1 subunit F, atp6v1f), SEQ ID NO: 10 The gene described (Cytochrome oxidase subunit I, COI), the gene described in SEQ ID NO: 11 (ATP citrate lyase isoform 2), the gene described in SEQ ID NO: 12 (Ribosomal protein L13a, rpl13a), the gene described in SEQ ID NO: 13 Cytochrome c oxidase subunit I), the gene described in SEQ ID NO: 14 (Pyrroline-5-carboxylate reductase 1, pycr1), the gene described in SEQ ID NO: 15 (Exs-related protein), the gene described in SEQ ID NO: 16 (Cysteine- rich with EGF-like domains 2, creld2), the gene described by SEQ ID NO: 17 (Selenoprotein 15), the gene described by SEQ ID NO: 18 (Beta-galactoside-binding lectin), the gene described by SEQ ID NO: 19 (hv1 gene for histone H2 variant), SEQ ID NO: 20 Gene (LAG1 longevity assurance 2), gene described in SEQ ID NO: 21 (Inositol oxygenase), gene described in SEQ ID NO: 22 (Acyl-CoA synthetase long-chain family member 1), gene described in SEQ ID NO: 23 Tetraspanin-3), the gene described by SEQ ID NO: 24 (Microsomal triglyceride transfer protein), the gene described by SEQ ID NO: 25 (Amino-terminal enhancer of split), the gene described by SEQ ID NO: 26 (non-classical MHC class I antigen , The gene described in SEQ ID NO: 27 (NADH-cytochrome b5 reductase 3), the gene described in SEQ ID NO: 28 (rRNA 2-O-methyltransferase fibrillarin), the gene described in SEQ ID NO: 29 (Vitellogenin II, vit-6), The gene described by SEQ ID NO: 30 (Choriogenin H minor), the gene described by SEQ ID NO: 31 (Adenylosuccinate synthetase isozyme 2), the gene described by SEQ ID NO: 32 (Mannose-P-dolichol utilization defect 1b, mpdu1b), SEQ ID NO: 33 to The gene described (RAB3 GTPase activating protein subunit 2 (non-catalytic)), the gene described by SEQ ID NO: 34 (Tax1 (human T-cell leukemia virus type I) binding protein 1b), the gene described by SEQ ID NO: 35 (40S ribosomal protein S24-like protein), gene described by SEQ ID NO: 36 (Exosome complex exonuclease RRP4), gene described by SEQ ID NO: 37 (Iduronate 2-sulfatase precursor), gene described by SEQ ID NO: 38 (Vitellogenin 1, ol- vit1), the gene set forth in SEQ ID NO: 41 (B-cell antigen receptor complex-associated protein alpha-chain precursor), the gene set forth in SEQ ID NO: 42 (Alpha-2,3-sialyltransferase ST3Gal I), set forth in SEQ ID NO: 43 Gene (Glutathione reductase (mitochondrial)), gene described by SEQ ID NO: 44 (Choriogenin L), gene described by SEQ ID NO: 45 (Integral membrane protein 1), gene described by SEQ ID NO: 46 (Complement control protein factor I- B), Gene described by SEQ ID NO: 47 (Glutamate receptor, ionotropic, delta 1, GRID1), gene described by SEQ ID NO: 48 (Dolichyl-alpha-1,6-mannosyltransferase (alg12)), gene described by SEQ ID NO: 49 (Macrosialin precursor), the gene described by SEQ ID NO: 50 (Metalloreductase STEAP4), the gene described by SEQ ID NO: 51 (14-alpha demethylase (CYP51)), and the gene described by SEQ ID NO: 52 (Bromodomain containing 2 (RING3)).

In the gene group, a gene whose expression is reduced in response to an estrogen-based steroid hormone may include, but is not limited to:

The gene described by SEQ ID NO: 1 (Dimethylglycine dehydrogenase), the gene described by SEQ ID NO: 2 (Fructose-bisphosphate aldolase B), the gene described by SEQ ID NO: 3 (Fatty acid binding protein 10 liver basic), described by SEQ ID NO: 4 Gene (Claudin), gene described by SEQ ID NO: 5 (Cytochrome P450 2P3), gene described by SEQ ID NO: 6 (Aldolase B), gene described by SEQ ID NO: 39 (Oligosaccharyltransferase complex subunit, ostc) and SEQ ID NO: 40 Gene (Abhydrolase domain containing 11).

The microarray chip may be an oligonucleotide or a complementary strand molecule thereof, which is a whole or a fragment thereof, of a nucleic acid sequence of a gene related to a self-defense mechanism against any one or more external stress selected from the following groups, but is not limited thereto. I never do that:

Gene described in SEQ ID NO: 5 (Cytochrome P450 1A, CYP1A), Gene described in SEQ ID NO: 8 (Selenoprotein M), Gene described in SEQ ID NO: 38 (Vitellogenin 1), Gene described in SEQ ID NO: 53 (Apolipoprotein B ), The gene described in SEQ ID NO: 54 (Glutamate dehydrogenase 1b), the gene described in SEQ ID NO: 55 (Glucose-6-phosphate dehydrogenase), and the gene described in SEQ ID NO: 56 (Transferrin).

The gene may be derived from an ocean scorpion (Oryzias javanicus), but is not limited thereto.

The estrogen-based steroid hormones may be 17β-estradiol (E2), estrone (estrone, E1), and estriol (estriol, E3), more specifically 17β-estradiol, but It is not limited.

The microarray chip may detect a reaction corresponding to 17β-estradiol contamination of the sea song, but is not limited thereto.

The present inventors exposed sea pods to 100 ㎍ / L of 17β-estradiol for 24 hours and 48 hours, respectively, in order to discover sea pod genes corresponding to 17β-estradiol exposure. The changing genes were examined. Specifically, cDNA was synthesized after each mRNA was isolated from sea trout (control) or sea trout (experimental) tissue cultured in seawater containing 17β-estradiol. PCR fragments were used to amplify gene fragments with altered expression levels. As a result, 56 genes with increased or decreased expression were selected in response to 17β-estradiol exposure. Estrogen-based steroid hormones, such as 17β-estradiol, inhibit the normal endocrine system function of males by inducing vitelogenin synthesis and femaleization in male fish, resulting in lowered fertility of males and disrupting ecosystems May result. Therefore, it is possible to confirm the stress and health status of marine organisms caused by external environmental changes by identifying changes in gene expression, which is the cause of physiological and metabolic changes, and to use these genes as biomarkers for diagnosing environmental and biological health. Can be.

Therefore, since the genes showed a significant change in gene expression in response to 17β-estradiol exposure, marker genes and microarray using the same for diagnosing stress and detecting the condition of persistent toxic substances such as 17β-estradiol. Can be usefully used as a chip

In addition,

1) isolating RNA from the sea pods of the experimental group treated with the test sample to determine whether the estrogen-based steroid hormone exposure and the sea pods of the control group treated with the test sample containing no steroid hormones of the estrogen series;

2) synthesizing cDNA from RNA of the experimental group and the control group of step 1) and labeling the experimental group and the control group with different fluorescent materials;

3) hybridizing cDNA labeled with different fluorescent materials of step 2) with the microarray chip;

4) analyzing the reacted microarray chip; And

5) provides a method for detecting the exposure of steroid hormones of the estrogen series and a diagnosis of environmental pollution, comprising the step of confirming the degree of gene expression integrated in the microarray chip in the analyzed data compared with the control group.

In the above method, the estrogen-based steroid hormone may be 17β-estradiol, but is not limited thereto.

In the method, the fluorescent material of step 2) is Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate ( rhodamine-B-isothiocyanate (RITC) and rhodamine may be selected from the group consisting of, but not limited to, any fluorescent material known to those skilled in the art can be used.

In the method, the microarray chip of step 4) can be used as long as the gene is loaded. All of the above procedures are preferably performed according to a general microarray chip experimental protocol, but are not limited thereto.

In addition,

1) isolating RNA from the sea pods of the experimental group treated with the test sample to determine whether the estrogen-based steroid hormone exposure and the sea pods of the control group treated with the test sample containing no steroid hormones of the estrogen series;

2) performing a real-time reverse transcript polymerase chain reaction (RT-PCR) using a pair of primers complementary to the gene and amplifying the RNA of step 1); And

3) provides a method for detecting the exposure to steroid hormones and environmental pollution diagnostic method comprising the step of confirming the expression level of the gene product of step 2) compared to the control.

In the above method, the primer pair of step 2) is complementary to the gene selected in the present invention, and any primer pair capable of amplifying the gene can be used.

The estrogen-based steroid hormones may be 17β-estradiol, estrone and estriol, and more specifically 17β-estradiol, but are not limited thereto.

The present inventors have identified 56 gene groups whose expression is increased or decreased in response to 17β-estradiol exposure, and it was found that the genes can be used to diagnose the condition of the environment following 17β-estradiol exposure. Therefore, the gene corresponding to 17β-estradiol exposure of the present invention can be usefully used for stress detection and environmental pollution diagnosis using a microarray chip or real-time RT-PCR.

In another aspect, the present invention provides a kit for detecting the exposure to steroid hormones of the estrogen-based and environmental pollution diagnosis kit comprising the microarray chip.

The kit may additionally include a sea trout.

The estrogen-based steroid hormones may be 17β-estradiol, estrone and estriol, and more specifically 17β-estradiol, but are not limited thereto.

The kit may further include any one selected from the group consisting of a fluorescent substance group consisting of a streptavidin-like phosphatease conjugate, a chemiluminescent substance, and a chemiluminescent substance However, the present invention is not limited thereto.

The kit may be any one selected from the group consisting of buffer solution used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, reaction reagent group consisting of dNTPs and rNTP (pre-mixed or separate feed type), label reagent, and washing buffer solution But is not limited thereto.

By identifying 56 groups of genes whose expression increases or decreases in response to 17β-estradiol exposure, the present inventors have found that the genes can be used to diagnose conditions of the environment corresponding to 17β-estradiol exposure. . Therefore, the gene corresponding to the 17β-estradiol exposure of the present invention can be usefully used as a kit for stress detection and environmental pollution diagnosis including a microarray chip.

In addition, the present invention provides a kit for detecting estrogen-based steroid hormone exposure and environmental pollution diagnosis kit comprising a primer pair complementary to a gene integrated in a microarray chip and amplifying the gene.

The primer pair is complementary to the gene, and both forward and reverse primer pairs designed to amplify the gene and designed for amplification products of 100 to 300 bp are available.

The kit may additionally include a sea trout.

The estrogen-based steroid hormones may be 17β-estradiol, estrone and estriol, and more specifically 17β-estradiol, but are not limited thereto.

The kit may further include any one or more selected from the group of reverse reagents for synthesizing cDNA from RNA, cNTPs and rNTP (premixed or separated feed), DNA polymerase, and a reaction buffer group. However, it is not limited thereto.

The present inventors have identified 56 gene groups whose expression is increased or decreased in response to 17β-estradiol exposure, and it was found that the genes can be used to diagnose the condition of the environment following 17β-estradiol exposure. Therefore, the present invention can be usefully used as a kit for detecting exposure to steroid hormones and environmental pollution diagnosis kit containing a primer pair for a gene corresponding to 17β-estradiol exposure of the present invention.

Hereinafter, the contents of the present invention will be described in more detail with reference to the following examples.

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

< Example  1> Sea trout  Incubation and 17β- Estradiol (17β- estradiol , E2 ) Exposure

<1-1> Sea trout  culture

Sea scallions were incubated in natural seawater through three types of filters (100, 10 and 1 μm). The water temperature was fixed to 25 ℃ using an underwater heater, and once a day Artemia Salina ( Artemia salina ) larvae were fed as food.

<1-2> 17β- Estradiol  exposure

In the culture tank, the males of the sea larvae were transferred to 3 L beakers containing 2 L of filtered seawater, and each was incubated for 24 hours. The 5 males of the sea squid males of Example <1-1> were exposed to 100 μg / L of 17β-estradiol for 24 hours and 48 hours, respectively. They moved one sea lion to ice water, instantly stunned them, then cut their heads, split their belly, and extracted the liver.

< Example  2> 17β- Estradiol  Genetic change measurement due to exposure

<2-1> RNA Separation of

1 ml of TRI Reagent solution (Molecular Research Center Inc) was added between the 17β-estradiol exposure test group (24 hours and 48 hours) obtained in Example <1-2> and the seafish of the unexposed control group, and the glass homogeneity. Homogenized using a glass homogenizer and allowed to stand at room temperature for 5 minutes. 200 μl of chloroform was added and mixed, and the mixture was left at room temperature for 10 minutes, and centrifuged at 12,000 × g and 4 ° C. for 15 minutes. The supernatant was obtained, 500 µl of isopropanol was added thereto, and left at room temperature for 5 minutes. After centrifugation at 12,000 × g, 4 ° C. for about 20 minutes, the solution was removed to give a precipitate. 50 μl of a 70% ethanol solution was added to the precipitate, followed by centrifugation for 5 minutes, the ethanol solution was removed, and the precipitated RNA was dried. After drying, it was dissolved in an appropriate amount of diethylpyrocarbonate (DEPC) -treated water.

<2-2> Cy3  And Cy5  sign

Total RNA from purified exposed and control groups was labeled with Cy3 and Cy5 using Agilent's Low RNA Input Linear Amplification Kit Plus (Agilent Technologies) as follows. Specifically, 1 μg of RNA was mixed with a dT-promoter primer and a MMLV-Reverse transcriptase, and reverse transcription was performed at 40 ° C. for 2 hours. Then, linear amplification was performed for 2 hours at 40 ° C. by adding T7 polymerase. Through the amplification process, the samples of the experimental group and the control group were labeled with Cy3-CTP and Cy5-CTP, respectively.

<2-3> Hybridization ( Hybridization ) And scanning ( scanning )

Fluorescent labeled cRNA samples were purified using Qiagen PCR purification kit and eluted with distilled water. Purified fluorescent label-cRNA samples were added to hybridization buffer (3x SSC, 0.3% SDS, 50% formamide, 20 μg Cot-1 DNA, 20 μg yeast tRNA). Concentration with microcon YM-30 gave a hybridization mixture. The hybridization mixture was heated to 95 ° C. for 3 minutes to denature and centrifuged at 12,000 × g for 30 seconds to cool the temperature. Coverslips were prepared on the prepared Sakura cDNA microarray, and the denatured hybridization mixture was pipetted. The microarray was placed in a GT-Hyb chamber (Chamber) and reacted at 65 ° C. for 16 hours. After the hybridization was completed, the microarray was removed from the chamber, the washing process was performed in the following order, and the microarray was rotated to dry and stored in the dark until scanning. The experimented seafish microarray was scanned using an Axon GenePix 4000B scanner (Axon Instrument). In a GenePix Pro 6.0 program, each point is drawn from a scanned image using a grid file and quantified to include a GPR file including analysis values such as Cy5 / Cy3 intensity and ratio of each point. Got.

<2-4> Microarray  Data analysis

From the GPR file obtained in the GenePix Pro 6.0 program, the analysis was performed using GeneSpring 7.3.1 (Agilent Technologies), an analysis program as follows. Normalization was performed using locally weighted regression scatterplot smoothing (LOWESS). Reliable genes were obtained by flag-out that the sum of the median values was lower than the background value or the standard deviation of each pixel value was not significant. As significant genes, spots with a difference in level value of 10 times or more were selected.

As a result, the gene expression was increased or decreased more than two times compared to the control by 17β-estradiol exposure during 24 hours (Table 1) and 48 hours (Table 2), of which 10 times or more 52 species showing changes are set forth in SEQ ID NOs: 1 to 52, and nucleic acid sequences for these are as follows.

Gene name Multiple increase SEQ ID NO: Down-regulation Dimethylglycine dehydrogenase 20.2 ± 0.01 One Fructose-bisphosphate aldolase B 14.3 ± 0.02 2 Fatty acid binding protein 10, liver basic 12.5 ± 0.08 3 Claudin 10.0 ± 0.02 4 Cytochrome P450 2P3 10.0 ± 0.03 5 Aldolase B 10.2 ± 0.05 6 Up-regulation Cytochrome c-1 (cyc1) 10.3 ± 0.03 7 Selenoprotein M 10.3 ± 0.03 8 ATPase, H + transporting, V1 subunit F (atp6v1f) 10.4 ± 0.03 9 Cytochrome oxidase subunit Ⅰ (COⅠ) gene 10.7 ± 0.06 10 Similar to ATP citrate lyase isoform 2 11.4 ± 0.03 11 Ribosomal protein L13a (rpl13a) 11.7 ± 0.09 12 Cytochrome c oxidase subunit Ⅰ 12.6 ± 0.45 13 Pyrroline-5-carboxylate reductase 1 (pycr1) 12.8 ± 0.23 14 Similar to Exs-related protein (LOC100150890) 13.0 ± 0.05 15 Cysteine-rich with EGF-like domains 2 (creld2) 13.2 ± 0.02 16 Selenoprotein 15 13.4 ± 0.03 17 Beta-galactoside-binding lectin 14.9 ± 0.01 18 hv1 gene for histone H2 variant 14.9 ± 0.05 19 Similar to LAG1 longevity assurance 2 17.1 ± 0.02 20 Inositol oxygenase 17.1 ± 0.06 21 Acyl-CoA synthetase long-chain family member 1 17.3 ± 0.23 22 Tetraspanin-3 (putative) 17.9 ± 0.01 23 Microsomal triglyceride transfer protein 18.1 ± 0.01 24 Amino-terminal enhancer of split (aes) 18.1 ± 0.09 25 non-classical MHC class I antigen (Orda-UDA) 18.2 ± 0.03 26 NADH-cytochrome b5 reductase 3 19.7 ± 0.06 27 rRNA 2-O-methyltransferase fibrillarin (fbrl) 20.5 ± 0.23 28 Vitellogenin Ⅱ (vit-6) 20.8 ± 0.13 29 Choriogenin H minor 21.5 ± 0.03 30 Adenylosuccinate synthetase isozyme 2 21.5 ± 0.18 31 Mannose-P-dolichol utilization defect 1b (mpdu1b) 21.6 ± 0.02 32 RAB3 GTPase activating protein subunit 2 (non-catalytic) 23.7 ± 0.03 33 Tax1 (human T-cell leukemia virus type I) binding protein 1b 25.0 ± 0.01 34 40S ribosomal protein S24-like protein 26.8 ± 0.02 35 Exosome complex exonuclease RRP4 35.2 ± 0.04 36 Similar to Iduronate 2-sulfatase precursor (ids) 36.8 ± 0.03 37 Vitellogenin 1 (ol-vit1) 53.1 ± 0.13 38

Gene name Multiple increase SEQ ID NO: Down-regulation Oligosaccharyltransferase complex subunit (ostc) 100.0 + - 0.01 39 Abhydrolase domain containing 11 11.1 ± 0.06 40 Fatty acid binding protein 10, liver basic 10.0 ± 0.04 3 Fructose-bisphosphate aldolase B 10.2 ± 0.01 2 Up-regulation B-cell antigen receptor complex-associated protein alpha-chain precursor 10.0 ± 0.05 41 Cytochrome oxidase subunit Ⅰ (COⅠ) 10.5 ± 0.01 10 Alpha-2,3-sialyltransferase ST3Gal Ⅰ 10.5 ± 0.07 42 Inositol oxygenase 11.5 ± 0.03 21 Glutathione reductase (mitochondrial) 11.7 ± 0.02 43 Pyrroline-5-carboxylate reductase 1 (pycr1) 11.8 ± 0.01 14 Choriogenin L 12.1 ± 0.02 44 Similar to integral membrane protein 1 12.3 ± 0.02 45 Complement control protein factor Ⅰ-B 12.5 ± 0.02 46 Glutamate receptor, ionotropic, delta 1 (GRID1) 12.7 ± 0.03 47 Dolichyl-alpha-1,6-mannosyltransferase (alg12) 12.7 ± 0.03 48 Macrosialin precursor 13.3 ± 0.02 49 hv1 gene for histone H2 variant 13.6 ± 0.06 19 Similar to ATP citrate lyase isoform 2 13.7 ± 0.01 11 Selenoprotein M 13.9 ± 0.01 8 40S ribosomal protein S24-like protein 14.6 ± 0.03 35 Metalloreductase STEAP4 14.7 ± 0.02 50 Vitellogenin Ⅱ (vit-6) 14.9 ± 0.01 29 NADH-cytochrome b5 reductase 3 16.4 ± 0.02 27 Similar to Exs-related protein (LOC100150890) 16.7 ± 0.05 15 RAB3 GTPase activating protein subunit 2 (non-catalytic) 17.2 ± 0.01 33 Tetraspanin-3 17.4 ± 0.08 23 Beta-galactoside-binding lectin 18.9 ± 0.02 18 Similar to LAG1 longevity assurance 2 19.3 ± 0.05 20 14-alpha demethylase (CYP51) 19.7 ± 0.04 51 Exosome complex exonuclease RRP4 21.7 ± 0.02 36 Tax1 (human T-cell leukemia virus type I) binding protein 1b 23.4 ± 0.01 34 Mannose-P-dolichol utilization defect 1b (mpdu1b) 23.6 ± 0.01 32 Choriogenin H minor 23.9 ± 0.02 30 Adenylosuccinate synthetase isozyme 2 27.8 ± 0.23 31 rRNA 2-O-methyltransferase fibrillarin (fbrl) 31.7 ± 0.12 28 Microsomal triglyceride transfer protein 37.1 ± 0.08 24 Acyl-CoA synthetase long-chain family member 1 40.1 ± 0.01 22 Bromodomain containing 2 (RING3) 45.2 ± 0.03 52 Similar to Iduronate 2-sulfatase precursor (ids) 49.1 ± 0.01 37 Vitellogenin 1 (ol-vit1) 59.1 ± 0.02 38

Example 3 Quantitative Analysis of Changes in Expression of Selected Genes

<3-1> Selection of genes related to self defense mechanism against external stress

Of the genes of sea larvae whose expression levels are more than two times isolated in Example <2-4>, the following seven genes, which are deeply related to the self-defense mechanism against external stress, were selected.

Apolipoprotein B, Cytochrome P450 1A, CYP1A, Glutamate dehydrogenase 1b, Glucose-6-phosphate dehydrogenase, Transferrin , Vitellogenin 1 and Selenoprotein M.

For these, primers for real-time quantitative PCR (qPCR) were designed and synthesized (Table 3). Changes in the expression levels of the genes were confirmed in liver samples of sea pods exposed to 17β-estradiol for 48 hours.

gene Base sequence of primer SEQ ID NO: Apofatty Protein B F: 5'-AAGCTAATGCCGAGGTTGATC-3 '
R: 5'-GGAGGCAGAGGACTTGAATG-3 ' 57
58 Cytochrome P450 1A (CYP1A) F: 5'-TCAACCAGTGGCAGATAAACC-3 '
R: 5'-CTGGCACTTCCTCAAATCTC-3 ' 59
60 Glutamate Dehydrogenase 1b F: 5'-CGATGCCAGGTCCGATGAAAC-3 '
R: 5'-GCCCAACACAGCCAGCACACAG-3 ' 61
52 Glucose-6-phosphate dehydrogenase F: 5'-AGGTGTCCCACTATCGGTTC-3 '
R: 5'-CTTCATCCTCCAGAGCTTGTG-3 ' 63
64 Transferrin F: 5'-CCTGGAATCTGACGACTACCA-3 '
R: 5'-CCTGGAATCTGACGACTACC-3 ' 65
66 Vittelogenin 1 F: 5'-ACAAAAGGTTCCACTCTCAGC-3 '
R: 5'-CCAACTTTAACCTCCATCTCC-3 ' 67
68 Selenium Protein M F: 5'-GGTCAAAGCTTTTGTGACTC-3 '
R: 5'-TCTGGCTTCTCCTTCTTGTAG-3 ' 69
70

<3- 2> cDNA synthesis

CDNA was synthesized using AB High Capacity RAN-to-cDNA Kit (Applied Biosystems, USA) using the RNA extracted by the method of Example <2-1> as a template. The amount corresponding to 1 RNA of RNA was dispensed and added to distilled water to make 9 ㎕. 1 μl of 2 × RT buffer 10 and 20 μl Enzyme Mix was added, mixed well, and precipitated by centrifugation. The mixture was reacted at 37 ° C. for 60 minutes. After the reaction, the reaction was terminated by heating at 95 ° C. for 5 minutes. The synthesized cDNA was stored at -20 ° C.

<3-3> Real-time quantitative PCR ( Real - time quantitative PCR ) analysis

0.5 μg (corresponding to about 10 ng / μl) of the synthesized cDNA, 0.8 μl (10 pmol / μl) of the primer pair of the gene was added, and 10 μl of the 2 SYBR1 mixture. The final amount was titrated to 20 증 with distilled water. 40 cycles of PCR were performed under the conditions of 95 ° C 10 minutes, 95 ° C 30 seconds, 60 ° C 30 seconds, and 72 ° C 30 seconds.

The CT values of the genes of interest (β-actin) and the gene of interest for each sample were compared, and relative expression curves of the gene of interest were quantified by a relative standard curve method (Table 4).

gene Exposure time SEQ ID NO:  24 hours Apofatty Protein B 2.5x reduction 53 Cytochrome P450 1A (CYP1A) (CYP1A) 2.7x reduction 5 Glutamate Dehydrogenase 1b 5.2x reduction 54 Glucose-6-phosphate dehydrogenase 2.2x reduction 55 Transferrin 3.6x reduction 56 Vittelogenin 1 59.1 times increase 37 Selenium Protein M 7.7x increase 8

The gene of the sea trellis corresponding to the 17β-estradiol (E2) exposure presented in the present invention is a 17β-estradiol exposure-specific gene, which is used to monitor and diagnose endocrine disruption and its environment. It can be usefully used as a biosensor for biomarkers, and can be used as biomarkers and sensors that can predict future pathological phenomena by specifying metabolic / physiological changes of the organism based on the functions of the genes presented. It can also be effectively used for stress source detection or environmental pollution diagnosis methods.

Attach an electronic file to a sequence list

Claims (20)

Microarray chip for detecting the exposure of 17β-estradiol (E2) in the sample, wherein the nucleic acid sequence of any one or more genes selected from the following groups are integrated:
The gene described by SEQ ID NO: 1 (Dimethylglycine dehydrogenase), the gene described by SEQ ID NO: 2 (Fructose-bisphosphate aldolase B), the gene described by SEQ ID NO: 3 (Fatty acid binding protein 10 liver basic), described by SEQ ID NO: 4 Gene (Claudin), gene described by SEQ ID NO: 5 (Cytochrome P450 2P3), gene described by SEQ ID NO: 6 (Aldolase B), gene described by SEQ ID NO: 7 (Cytochrome c-1, cyc1), SEQ ID NO: 8 The gene described (Selenoprotein M), the gene described in SEQ ID NO: 9 (ATPase H + transporting V1 subunit F, atp6v1f), the gene described in SEQ ID NO: 10 (Cytochrome oxidase subunit I, COI), the gene described in SEQ ID NO: 11 ATP citrate lyase isoform 2), the gene described by SEQ ID NO: 12 (Ribosomal protein L13a, rpl13a), the gene described by SEQ ID NO: 13 (Cytochrome c oxidase subunit I), the gene described by SEQ ID NO: 14 (Pyrroline-5-carboxylate red uctase 1, pycr1), the gene described in SEQ ID NO: 15 (Exs-related protein), the gene described in SEQ ID NO: 16 (Cysteine-rich with EGF-like domains 2, creld2), the gene described in SEQ ID NO: 17 (Selenoprotein 15), the gene described by SEQ ID NO: 18 (Beta-galactoside-binding lectin), the gene described by SEQ ID NO: 19 (hv1 gene for histone H2 variant), the gene described by SEQ ID NO: 20 (LAG1 longevity assurance 2), sequence The gene described in SEQ ID NO: 21 (Inositol oxygenase), the gene described in SEQ ID NO: 22 (Acyl-CoA synthetase long-chain family member 1), the gene described in SEQ ID NO: 23 (Tetraspanin-3), described in SEQ ID NO: 24 Microsomal triglyceride transfer protein, gene described by SEQ ID NO: 25 (Amino-terminal enhancer of split), gene described by SEQ ID NO: 26 (non-classical MHC class I antigen), gene described by SEQ ID NO: 27 (NADH -cytochrome b5 reductase 3), sequence The gene described in SEQ ID NO: 28 (rRNA 2-O-methyltransferase fibrillarin), the gene described in SEQ ID NO: 31 (Adenylosuccinate synthetase isozyme 2), the gene described in SEQ ID NO: 32 (Mannose-P-dolichol utilization defect 1b, mpdu1b), The gene described in SEQ ID NO: 33 (RAB3 GTPase activating protein subunit 2 (non-catalytic)), the gene described in SEQ ID NO: 34 (Tax1 (human T-cell leukemia virus type I) binding protein 1b), described in SEQ ID NO: 35 Gene (40S ribosomal protein S24-like protein), gene described by SEQ ID NO: 36 (Exosome complex exonuclease RRP4), gene described by SEQ ID NO: 37 (Iduronate 2-sulfatase precursor), gene described by SEQ ID NO: 39 (Oligosaccharyltransferase complex subunit (ostc), the gene represented by SEQ ID NO: 40 (Abhydrolase domain containing 11), the gene described by SEQ ID NO: 41 (B-cell antigen receptor complex-associated protein alpha-chain precursor), SEQ ID NO: The gene described in 42 (Alpha-2,3-sialyltransferase ST3Gal I), the gene described in SEQ ID NO: 43 (Glutathione reductase (mitochondrial)), the gene described in SEQ ID NO: 45 (Integral membrane protein 1), SEQ ID NO: 46 Gene described (Complement control protein factor I-B), gene represented by SEQ ID NO: 47 (Glutamate receptor, ionotropic, delta 1, GRID1), gene described by SEQ ID NO: 48 (Dolichyl-alpha-1,6-mannosyltransferase ( alg12)), the gene described in SEQ ID NO: 49 (Macrosialin precursor), the gene described in SEQ ID NO: 50 (Metalloreductase STEAP4), the gene described in SEQ ID NO: 51 (14-alpha demethylase (CYP51)) described in SEQ ID NO: 52 Gene (Bromodomain containing 2 (RING3)), gene described by SEQ ID NO: 53 (Apolipoprotein B), gene described by SEQ ID NO: 54 (Glutamate dehydrogenase 1b), gene described by SEQ ID NO: 55 (Glucose-6-phosphate dehydrogenase) Gene described in SEQ ID NO: 56 (Transferrin).
The microarray chip of claim 1, wherein the gene is derived from Oryzias javanicus .
delete According to claim 1, wherein the gene described in SEQ ID NO: 7 (Cytochrome c-1, cyc1), the gene described in SEQ ID NO: 8 (Selenoprotein M), the gene described in SEQ ID NO: 9 (ATPase H + transporting V1 subunit F, atp6v1f ), The gene described in SEQ ID NO: 10 (Cytochrome oxidase subunit I, COI), the gene described in SEQ ID NO: 11 (ATP citrate lyase isoform 2), the gene described in SEQ ID NO: 12 (Ribosomal protein L13a, rpl13a), SEQ ID NO: 13, gene (Pyrroline-5-carboxylate reductase 1, pycr1), gene represented by SEQ ID NO: 15 (Exs-related protein), SEQ ID NO: 16 Gene described (Cysteine-rich with EGF-like domains 2, creld2), gene described by SEQ ID NO: 17 (Selenoprotein 15), gene described by SEQ ID NO: 18 (Beta-galactoside-binding lectin), described by SEQ ID NO: 19 Hv1 gene for histone H2 variant, The gene described by SEQ ID NO: 20 (LAG1 longevity assurance 2), the gene described by SEQ ID NO: 21 (Inositol oxygenase), the gene described by SEQ ID NO: 22 (Acyl-CoA synthetase long-chain family member 1), SEQ ID NO: 23 The gene described (Tetraspanin-3), the gene described in SEQ ID NO: 24 (Microsomal triglyceride transfer protein), the gene described in SEQ ID NO: 25 (Amino-terminal enhancer of split), the gene described in SEQ ID NO: 26 (non-classical MHC class I antigen, gene described by SEQ ID NO: 27 (NADH-cytochrome b5 reductase 3), gene described by SEQ ID NO: 28 (rRNA 2-O-methyltransferase fibrillarin), gene described by SEQ ID NO: 31 (Adenylosuccinate synthetase isozyme 2 ), The gene described in SEQ ID NO: 32 (Mannose-P-dolichol utilization defect 1b, mpdu1b), the gene described in SEQ ID NO: 33 (RAB3 GTPase activating protein subunit 2 (non-catalytic)), described in SEQ ID NO: 34 Gene (Tax1 (human T-cell leukemia virus type I) binding protein 1b), gene represented by SEQ ID NO: 35 (40S ribosomal protein S24-like protein), gene described by SEQ ID NO: 36 (Exosome complex exonuclease RRP4), sequence Gene described as ID 37 (Iduronate 2-sulfatase precursor), Gene described as SEQ ID NO: 41 (B-cell antigen receptor complex-associated protein alpha-chain precursor), Gene described as SEQ ID NO: 42 (Alpha-2,3 -sylyltransferase ST3Gal I), the gene described in SEQ ID NO: 43 (Glutathione reductase (mitochondrial)), the gene described in SEQ ID NO: 45 (Integral membrane protein 1), the gene described in SEQ ID NO: 46 (Complement control protein factor I-B ), The gene described in SEQ ID NO: 47 (Glutamate receptor, ionotropic, delta 1, GRID1), the gene described in SEQ ID NO: 48 (Dolichyl-alpha-1,6-mannosyltransferase (alg12)), the gene described in SEQ ID NO: 49 (Macrosialin pre cursor), the gene described by SEQ ID NO: 50 (Metalloreductase STEAP4), the gene described by SEQ ID NO: 51 (14-alpha demethylase (CYP51)), and the gene described by SEQ ID NO: 52 (Bromodomain containing 2 (RING3)) are 17β- 17β-estradiol exposure detection microarray chip characterized in that the expression increases in response to estradiol exposure.
The method of claim 1, wherein the gene of SEQ ID NO: 1 (Dimethylglycine dehydrogenase), the gene of SEQ ID NO: 2 (Fructose-bisphosphate aldolase B), the gene of SEQ ID NO: 3 (Fatty acid binding protein 10 liver basic), Gene described by SEQ ID NO: 4 (Claudin), Gene described by SEQ ID NO: 5 (Cytochrome P450 2P3), Gene described by SEQ ID NO: 6 (Aldolase B), Gene described by SEQ ID NO: 39 (Oligosaccharyltransferase complex subunit, ostc) And the gene described in SEQ ID NO: 40 (Abhydrolase domain containing 11) has a decrease in expression in response to 17β-estradiol exposure, microarray chip for detecting the exposure to 17β-estradiol.
The micro-array chip of claim 1, wherein the microarray chip is integrated with a nucleic acid sequence of a gene related to a self-defense mechanism against any one or more external stresses selected from the following group. 17. Array chip:
Gene described in SEQ ID NO: 5 (Cytochrome P450 1A, CYP1A), Gene described in SEQ ID NO: 8 (Selenoprotein M), Gene described in SEQ ID NO: 53 (Apolipoprotein B), Gene described in SEQ ID NO: 54 (Glutamate dehydrogenase 1b), the gene described by SEQ ID NO: 55 (Glucose-6-phosphate dehydrogenase) and the gene described by SEQ ID NO: 56 (Transferrin).
1) isolating RNA from the sea pods of the experimental group treated with the test sample to determine whether the 17β- estradiol exposure and the sea pods of the control group treated with the test sample containing no 17β- estradiol;
2) synthesizing cDNA from RNA of the experimental group and the control group of step 1) and labeling the experimental group and the control group with different fluorescent materials;
3) hybridizing the cDNA labeled with different fluorescent substances of step 2) with the microarray chip of claim 1;
4) analyzing the reacted microarray chip; And
5) detecting the expression level of 17β-estradiol in the sample, comprising the step of confirming the degree of gene expression integrated in the microarray chip of claim 1 in the analyzed data compared to the control.
The method of claim 7, wherein the sample is any one selected from the group consisting of a biological sample, a food sample, a chemical sample, an industrial sample, a clinical sample, and an environmental sample.
delete The method of claim 7, wherein the phosphor of step 2) is Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (rhodamine-B-isothiocyanate, RITC) and rhodamine (rhodamine) is selected from the group consisting of 17β- estradiol exposure detection method.
1) separating RNA from sea pods of the experimental group treated with the test sample to be exposed to 17β-estradiol and sea pods of the control group treated with the test sample containing no 17β-estradiol;
2) performing a real-time reverse transcript polymerase chain reaction (RT-PCR) using a pair of primers complementary to the gene of claim 1 and amplifying the RNA of step 1); And
3) detecting the expression level of 17β-estradiol in the sample, comprising the step of comparing the gene product of step 2) with the control to confirm the expression level.
12. The method of claim 11, wherein the sample is any one selected from the group consisting of a biological sample, a food sample, a chemical sample, an industrial sample, a clinical sample, and an environmental sample.
delete Claim 1 kit comprising a microarray chip, 17β- estradiol exposure detection kit.
delete 15. The method of claim 14, further comprising any one selected from the group of fluorescent substances consisting of streptadin-like phosphatease conjugate, chemilurorensce and chemiluminescent. 17β-estradiol exposure kit for detecting, characterized in that.
15. The method according to claim 14, which is selected from the group of reaction reagents consisting of a buffer used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, dNTPs and rNTP (premixed or separated feed), a marker reagent, and a wash buffer. 17β-estradiol exposure detection kit, characterized in that it further comprises any one.
Comprising the gene of claim 1 and comprising a primer pair capable of amplifying the gene, 17β-estradiol exposure kit for detecting.
delete 19. The method of claim 18, further comprising at least one selected from the group consisting of reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (premixed or separated feed), DNA polymerase, and a reaction buffer group. 17β-estradiol exposure kit for detecting, characterized in that.