KR101199440B1 - Polycyclic aromatic hydrocarbons exposure responsive genes in Scleronephthya gracillimum and the method for diagnosing the coastal environment pollution using the same - Google Patents

Abstract

The present invention relates to a gene of Scleronephthya gracillimum corresponding to the exposure of polycyclic aromatic hydrocarbons and a method for diagnosing coastal environmental pollution using the same, and more particularly, to a specific gene group derived from the pink seamandrami. By expressing specific expression changes by this polycyclic aromatic hydrocarbon exposure and introducing them as biomarkers, it can be usefully used for detecting the stress of marine ecosystems following polycyclic aromatic hydrocarbon exposure and monitoring and determining the contamination of polycyclic aromatic hydrocarbons in environmental samples. It can be used as a tool to identify mechanisms of toxicity and carcinogenic effects caused by polycyclic aromatic hydrocarbons.

Description

Polycyclic aromatic hydrocarbons exposure responsive genes in Scleronephthya gracillimum and the method for diagnosing the coastal environment pollution using the same}

The present invention relates to a gene of Scleronephthya gracillimum corresponding to the exposure of polycyclic aromatic hydrocarbons (PAHs) and a coastal environmental pollution diagnosis method using the same.

Scleronephthya gracillimum ) is a type of arithmetic lake that widely inhabits the coastal waters of Jeju, especially the southern part of Seogwipo, and is distributed from southern Japan to the East China Sea. The colony is very elastic, and when it is unfolded, it is shrub-like, but when it contracts, it turns into a lumpy mass. On the surface of the disease, there are transverse wrinkles, and in the tube, the highly contractive polyps are gathered in a lump form. The body is light yellow, red yellow, orange or pink, and the polyp mass is pink or orange. Lives in groups on vertical walls or heavily sloped rocks, distributed about 3-4 m deep and 15 m deep. By providing habitats for other creatures, it is a creature that makes a great contribution to Korea's marine biodiversity. Therefore, in order to preserve the marine biodiversity of the coast of Jeju, it is necessary to understand the response of the species to the anticipated coastal pollutants, and to diagnose the species or diagnose the coastal pollution based on this species.

Polycyclic aromatic hydrocarbons (PAHs) are aromatic hydrocarbons with multiple benzene rings and consist of many classes of compounds. It is produced by incomplete combustion of fossil fuels or industrial activities and is a major source of pollution to the environment and human body. Some of the major polycyclic aromatic hydrocarbons are classified as carcinogens by the specific pollutant carcinogenic classification (IARC) for human risk assessment because of their toxicity. . Long-term chronic exposure is considered more important than acute exposure as an environmental carcinogen.

Large quantities of polycyclic aromatic hydrocarbons are detected worldwide in freshwater and marine sediments in industrialized regions. Polycyclic aromatic hydrocarbons are more soluble in organic solvents than water, and are absorbed by aquatic organisms in an aquatic environment, or they are adsorbed to suspended matter and settle to the bottom of the water, accumulating on low soils, causing mutations in benthic organisms or killing them. (Varanasi et al . , Environ . Health Perspect . , 90, 93-100, 1991).

To counter these contaminants, marine organisms actively cope by changing the physiology or metabolism by controlling the expression level of their specific genes. Therefore, by discovering genes whose expression levels change due to exposure to pollutants such as polycyclic aromatic hydrocarbons and using them as biomarkers, exposure to such pollutants as well as information on exposure to polycyclic aromatic hydrocarbons in specific regions is vital. Information on the effects on the phenomena and the health of the organism can be obtained. These techniques can detect exposure to fine concentrations of contaminants that cannot be detected by analytical chemistry, have early diagnostic value, and allow for future predictive functions through consideration of gene function. Therefore, in order to understand and respond to the rapid global environmental changes caused by human activities, the necessity of developing advanced technologies such as diagnosing the effects of environmental changes and the ecosystem ripple effect through the analysis of genes and nucleic acid materials, which are the basics of life phenomena, is urgently needed. Do.

Thus, the inventors of the present invention, while discovering biomarkers corresponding to polycyclic aromatic hydrocarbon exposure, Scleronephthya , which is a kind of soft coral that plays a large role in maintaining marine biodiversity. gracillimum ) identified changes in the expression of specific genes due to polycyclic aromatic hydrocarbon exposure, and the specific gene candidate group corresponding to polycyclic aromatic hydrocarbon exposure is a biosensor that can grasp the degree of environmental change and the state of marine ecosystem according to pollutants. The present invention has been completed by suggesting that it can be usefully used.

It is an object of the present invention to counteract the exposure to polycyclic aromatic hydrocarbons ( Scleronephthya) gracillimum ) genes, and methods for detecting and diagnosing stress in coastal ecosystems against environmental pollution.

In order to achieve the above object, the present invention provides an oligonucleotide comprising all or part of a gene sequence whose expression is increased or decreased in response to exposure to polycyclic aromatic hydrocarbons, or oligonucleotide complementary to the oligonucleotide The present invention provides a microarray chip for stress detection and health diagnosis of marine ecosystems following exposure to polytoxic aromatic hydrocarbons with high levels of molecularly integrated genotoxicity.

In addition, the present invention provides a method for detecting stress and health diagnosis of marine ecosystems by exposure to polycyclic aromatic hydrocarbons of genotoxicity induced levels using the gene.

In addition, the present invention provides a kit for stress detection and health diagnostics of marine ecosystems following exposure to genotoxicity-induced polycyclic aromatic hydrocarbons, including the microarray chip.

In addition, the present invention provides a kit for stress detection and health diagnosis of marine ecosystems following exposure to genotoxicity induced polycyclic aromatic hydrocarbons, comprising primer pairs complementary to the gene and capable of amplifying the gene. .

By measuring the expression level of a specific gene group of the pink sea mandrami that exhibits a change in the expression amount by polycyclic aromatic hydrocarbon exposure, the specific gene of the pink sea mandrami corresponding to the polycyclic aromatic hydrocarbon exposure of the present invention is determined by the exposure to contaminants. As well as environmental changes, it can be usefully used as a biosensor and a diagnostic method for diagnosing stress and health of marine ecosystems.

1 is a diagram showing the difference in the gene expression of pink seawater Dramid cultured in filtered seawater or polycyclic aromatic hydrocarbon containing seawater (100 ppb) at 22 ℃.

Hereinafter, the present invention will be described in detail.

The present invention provides genes for stress detection and health diagnosis of marine ecosystems following exposure to polycyclic aromatic hydrocarbons at genotoxic induction levels, which are composed of the following groups:

Gene described in SEQ ID NO: 1 (calmodulin), gene described in SEQ ID NO: 2 (cytosolic malate dehydrogenase), gene described in SEQ ID NO: 3 (40S ribosomal protein S23), gene described in SEQ ID NO: 4 (protein disulfide isomerase) , A gene described in SEQ ID NO: 5 (dipeptidyl aminopeptidase IV), a gene described in SEQ ID NO: 6 (proteasome beta 3 subunit), a gene described in SEQ ID NO: 7 (ATP synthase F0 subunit 6), a gene described in SEQ ID NO: 8 (40S ribosomal protein S8), the gene described in SEQ ID NO: 9 (myosin heavy chain), the gene described in SEQ ID NO: 10 (MAK10 homolog, amino-acid N-acetyltransferase subunit), the gene described in SEQ ID NO: 11 (heletron 5 helitron-like transposon replicase / helicase / endonuclease, the gene described in SEQ ID NO: 12 (ral guanine nucleotide dissociation stimulator-like 1), the gene described in SEQ ID NO: 13 (allene oxide synthase / 8R-lipo xygenase fusion protein), gene described by SEQ ID NO: 14 (triacylglycerol lipase-like protein), gene described by SEQ ID NO: 15 (bromodomain adjacent to zinc finger domain 1A), gene described by SEQ ID NO: 16 (60S ribosomal protein L6) A gene described by SEQ ID NO: 17 (short-chain dehydrogenase / reductase 9), a gene described by SEQ ID NO: 18 (cardiac troponin C), a gene described by SEQ ID NO: 19 (calcium dependent mitochondrial carrier protein), SEQ ID NO: 20 To the gene described (mannose-binding lectin 2), gene represented by SEQ ID NO: 21 {CXXC finger 1 (PHD domain), isoform}, gene represented by SEQ ID NO: 22 (myosin light chain kinase isoform 3A), SEQ ID NO: 23 Gene described (transcriptional regulator, XRE family), gene described in SEQ ID NO: 24 (ubiquitin-ptotein ligase), gene described in SEQ ID NO: 25 (astacin protease 4), gene described in SEQ ID NO: 26 { poly (ADP-ribose) polymerase 4}, gene described by SEQ ID NO: 27 {ADAM metallopeptidase domain 28 (ADAM28)}, gene described by SEQ ID NO: 28 (Collagen, type XXV), gene described by SEQ ID NO: 29 (eukaryotic translation initiation factor 4 gamma, the gene described in SEQ ID NO: 30 (peptidylglycine alpha-hydroxylating monooxygenase), the gene described in SEQ ID NO: 31 (Ras-related and estrogen-regulated growth inhibitor), and the gene described in SEQ ID NO: 32 (small acid-soluble spore protein).

Genes whose expression increases in response to polycyclic aromatic hydrocarbon exposure in the gene group preferably include the following genes, but are not limited thereto:

Gene described in SEQ ID NO: 1 (calmodulin), gene described in SEQ ID NO: 2 (cytosolic malate dehydrogenase), gene described in SEQ ID NO: 3 (40S ribosomal protein S23), gene described in SEQ ID NO: 4 (protein disulfide isomerase) , A gene described in SEQ ID NO: 5 (dipeptidyl aminopeptidase IV), a gene described in SEQ ID NO: 6 (proteasome beta 3 subunit), a gene described in SEQ ID NO: 7 (ATP synthase F0 subunit 6), a gene described in SEQ ID NO: 8 (40S ribosomal protein S8), the gene described in SEQ ID NO: 9 (myosin heavy chain), the gene described in SEQ ID NO: 10 (MAK10 homolog, amino-acid N-acetyltransferase subunit), the gene described in SEQ ID NO: 11 (heletron 5 helitron-like transposon replicase / helicase / endonuclease, the gene described in SEQ ID NO: 12 (ral guanine nucleotide dissociation stimulator-like 1), the gene described in SEQ ID NO: 13 (allene oxide synthase / 8R-lipo xygenase fusion protein), gene described by SEQ ID NO: 14 (triacylglycerol lipase-like protein), gene described by SEQ ID NO: 15 (bromodomain adjacent to zinc finger domain 1A), gene described by SEQ ID NO: 16 (60S ribosomal protein L6) , Genes set forth in SEQ ID NO: 17 (short-chain dehydrogenase / reductase 9) and genes set forth in SEQ ID NO: 18 (cardiac troponin C).

Genes whose expression decreases in response to polycyclic aromatic hydrocarbon exposure in the gene group preferably include but are not limited to the following genes:

The gene described in SEQ ID NO: 19 (calcium dependent mitochondrial carrier protein), the gene described in SEQ ID NO: 20 (mannose-binding lectin 2), the gene described in SEQ ID NO: {CXXC finger 1 (PHD domain), isoform}, Myosin light chain kinase isoform 3A, SEQ ID NO: 22, Transcriptional regulator (XRE family), SEQ ID NO: 24 (ubiquitin-ptotein ligase), SEQ ID NO: 25 Gene (astacin protease 4), gene described by SEQ ID NO: 26 {poly (ADP-ribose) polymerase 4}, gene described by SEQ ID NO: 27 {ADAM metallopeptidase domain 28 (ADAM28)}, gene described by SEQ ID NO: 28 (Collagen, type XXV), the gene described in SEQ ID NO: 29 (eukaryotic translation initiation factor 4 gamma), the gene described in SEQ ID NO: 30 (peptidylglycine alpha-hydroxylating monooxygenase), the gene described in SEQ ID NO: 31 Here (Ras-related and estrogen-regulated growth inhibitor) and SEQ ID NO: gene described in 32 (small acid-soluble spore protein).

The gene described in SEQ ID NO: 1 (calmodulin), the gene described in SEQ ID NO: 18 (cardiac troponin C), and the gene described in SEQ ID NO: 19 (calcium dependent mitochondrial carrier protein) is a calcium binding motif (calcium binding motif) It is an EF-Hand superfamily protein gene having, the gene described in SEQ ID NO: 3 (40S ribosomal protein S23), the gene described in SEQ ID NO: 8 (40S ribosomal protein S8), and the gene described in SEQ ID NO: 16 (60S ribosomal protein L6) is a gene of ribosomal protein, preferably, but not limited to, contributing to structural stabilization by binding to rRNA during biosynthesis of members of ribosomes and proteins.

According to the functional classification of the gene based on the eukaryotic orthologous groups (KOG), the gene described in SEQ ID NO: 3 (40S ribosomal protein S23), the gene described in SEQ ID NO: 8 (40S ribosomal protein S8 ), The gene described in SEQ ID NO: 16 (60S ribosomal protein L6), the gene described in SEQ ID NO: 23 (transcriptional regulator, XRE family), and the gene described in SEQ ID NO: 29 (eukaryotic translation initiation factor 4 gamma) A gene related to ribosomal structure and biosynthesis, the gene described in SEQ ID NO: 24 (ubiquitin-ptotein ligase) is a gene involved in RNA processing and modification, and the gene described in SEQ ID NO: 21 {CXXC finger 1 (PHD domain) , isoform} and the gene described in SEQ ID NO: 26 {poly (ADP-ribose) polymerase 4} is a gene involved in transcription, the gene described in SEQ ID NO: 15 (bromodomain adjacent to zinc finger domain 1A) is a gene related to chromatin structure and dynamics, and the gene described in SEQ ID NO: 11 (heletron 5 helitron-like transposon replicase / helicase / endonuclease) is a cell cycle regulator, cell division, chromosome A gene related to the fraction, the gene described in SEQ ID NO: 1 (calmodulin), the gene described in SEQ ID NO: 12 (ral guanine nucleotide dissociation stimulator-like 1), the gene described in SEQ ID NO: 14 (triacylglycerol lipase-like protein) , The gene described by SEQ ID NO: 18 (cardiac troponin C), the gene described by SEQ ID NO: 19 (calcium dependent mitochondrial carrier protein), and the gene described by SEQ ID NO: 32 (small acid-soluble spore protein) A gene related to the gene (mannose-binding lectin 2) described in SEQ ID NO: 20 is a gene related to a defense mechanism; The gene described (protein disulfide isomerase), the gene described in SEQ ID NO: 5 (dipeptidyl aminopeptidase IV), the gene described in SEQ ID NO: 6 (proteasome beta 3 subunit), the gene described in SEQ ID NO: 27 (ADAM metallopeptidase domain 28 (ADAM28) )}, The gene described in SEQ ID NO: 30 (peptidylglycine alpha-hydroxylating monooxygenase) is a gene related to modification, turnover, and chaperone after protein synthesis, the gene described in SEQ ID NO: 9 (myosin heavy chain), And the gene described in SEQ ID NO: 22 (myosin light chain kinase isoform 3A) is a gene related to the cytoskeleton, and the gene described in SEQ ID NO: 28 (Collagen, type VII V) is a gene related to extracellular structure, The gene described as 2 (cytosolic malate dehydrogenase) and the gene described as SEQ ID NO: 7 (ATP synthase F0 subunit 6) are involved in energy production and conversion. The former, the gene described in SEQ ID NO: 13 (allene oxide synthase / 8R-lipoxygenase fusion protein) is a gene involved in carbohydrate transport and metabolism, the gene described in SEQ ID NO: 17 (short-chain dehydrogenase / reductase 9) is Genes involved in secondary metabolite biosynthesis, transport, and catabolism, genes described in SEQ ID NO: 10 (MAK10 homolog, amino-acid N-acetyltransferase subunit), genes described in SEQ ID NO: 25 (astacin protease 4), and The gene described in SEQ ID NO: 31 (Ras-related and estrogen-regulated growth inhibitor) is preferably a gene related to general function prediction, but is not limited thereto.

The gene is Scleronephthya gracillimum ) is preferably, but not limited to.

The inventors of the present invention synthesized cDNA of pink sea mandrami after culturing pink sea mandrami in polycyclic aromatic hydrocarbon standard reagent (Supelco, USA, 100 ppb) for 24 hours in order to discover the pink sea mandra gene corresponding to polycyclic aromatic hydrocarbon exposure. Genes whose expression levels change were examined. Specifically, after separating the mRNA from the pink seamandrami (control group) cultured in seawater at the same temperature as the control group containing a pink seamandrami (control group) or polycyclic aromatic hydrocarbons (22%), GeneFishing CDNA was synthesized from the purified mRNA using the ^TM DEG kit (Seegene, Korea). PCR fragments were used to amplify gene fragments with altered expression levels. The amplified cDNA fragments were cloned into T-vectors, plasmids were extracted, and sequencing was performed. As a result, 32 genes whose expression was increased or decreased by polycyclic aromatic hydrocarbon exposure were selected. Persistent organic toxicants, such as polycyclic aromatic hydrocarbons, cause toxic reactions in various aspects to organisms, and marine organisms cause changes in physiology and metabolism due to such environmental pollution. Therefore, it is possible to confirm the stress and health status of marine organisms according to the change of external environment by confirming the change of gene expression which is the cause of physiological and metabolic changes, and these genes are biomarkers that can diagnose the health of marine environment and marine organisms. Available as

Therefore, since the genes have been changed in response to polycyclic aromatic hydrocarbon exposure, they are useful as marker genes for diagnosing stress and detecting the condition of marine ecosystems caused by persistent organic toxic substances such as polycyclic aromatic hydrocarbons in coastal waters. Can be used.

In addition, the present invention is a stress detection of marine ecosystem in which cDNA fragment, oligonucleotide, gene fragment complementary to the cDNA fragment, or oligonucleotide molecule complementary to the oligonucleotide is integrated, including all or part of the nucleotide sequence of the gene And a microarray chip for medical examination.

The microarray chip preferably detects a marine ecosystem response corresponding to polycyclic aromatic hydrocarbon contamination at the level of genotoxic induction of pink sea cockscomb.

By identifying 32 gene groups whose expression increases or decreases in response to polycyclic aromatic hydrocarbon exposure, the present inventors can diagnose the state of the marine ecosystem due to polycyclic aromatic hydrocarbon contamination, which is a persistent organic toxic pollutant. And it was found. Therefore, the genes corresponding to the exposure of the persistent organic toxic contaminants of the present invention can be usefully used for microarray chips for stress detection and health diagnosis.

In addition, the present invention provides a method for detecting stress and health diagnosis of marine ecosystems by exposure to polycyclic aromatic hydrocarbons of genotoxicity induced levels using the gene.

Specifically, the method

1) separating the RNA from the experimental group pink sea cockscomb and the control pink sea cockscomb, respectively;

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 cDNAs labeled with different fluorescent materials of step 2) with an oligonucleotide comprising all or a part of the gene sequence or a microarray chip in which complementary oligonucleotides are integrated;

4) analyzing the reacted microarray chip of step 3); And

5) Preferably, but not limited to, including the step of confirming the expression level of the gene of the present invention compared to the control in the analyzed data of step 4).

In the above method, the experimental group of step 1) is a pink sea mandrami treated with seawater to confirm whether the polycyclic aromatic hydrocarbon exposure, and the control group is a pink sea mandrami treated seawater not containing polycyclic aromatic hydrocarbon, but It is not limited.

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

1) separating the RNA from the experimental group pink sea cockscomb and the control pink sea cockscomb, respectively;

2) performing a real-time reverse transcript polymerase chain reaction (qRT-PCR) using the RNA pair of step 1) using a primer pair complementary to the gene of the present invention and capable of amplifying the gene. ; And

3) It is preferable to include the step of confirming the expression level by comparing the gene product of step 2) with the control, but is not limited thereto.

In the above method, the experimental group of step 1) is a pink sea mandrami treated with seawater to confirm whether the polycyclic aromatic hydrocarbon exposure, and the control group is a pink sea mandrami treated seawater not containing polycyclic aromatic hydrocarbon, but It is not limited.

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 inventors have found that by identifying 32 gene groups whose expression increases or decreases in response to polycyclic aromatic hydrocarbon exposure, the genes can be used to diagnose the state of the marine ecosystem following polycyclic aromatic hydrocarbon exposure. Therefore, the gene corresponding to the polycyclic aromatic hydrocarbon exposure of the present invention can be usefully used for stress detection and health diagnosis of marine ecosystems using microarray chips or real-time RT-PCR.

In addition, the present invention provides a kit for stress detection and health diagnostics of the marine ecosystem including the microarray chip.

The kit preferably detects the marine ecosystem response corresponding to polycyclic aromatic hydrocarbon exposure of the genotoxic inducement level of pink sea cockscomb, but is not limited thereto.

The kit may further comprise a pink sea cockscomb.

The kit may additionally comprise a fluorescent material, wherein the fluorescent material is selected from the group consisting of streptavidin-alkaline phosphatase conjugates, chemilurorenscents and chemiluminescents. Preferably, but not limited to.

The kit may additionally include a reaction reagent, which is a buffer used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, dNTPs and rNTP (premixed or separated feed), chemical inducer of fluorescent dyes It may be composed of a labeling reagent, such as a washing buffer, but is not limited thereto, and any reaction reagents required for hybridization reactions known to those skilled in the art may be included.

The inventors have identified 32 gene groups whose expression is increased or decreased in response to polycyclic aromatic hydrocarbon exposure, and thus it was found that the genes can be used to diagnose the state of the marine ecosystem according to the change in polycyclic aromatic hydrocarbon exposure. . Therefore, the gene corresponding to the polycyclic aromatic hydrocarbon exposure of the present invention can be usefully used as a stress detection and health diagnostic kit of the marine ecosystem including the microarray chip.

In addition, the present invention provides a kit for stress detection and health diagnostics of marine ecosystems comprising primer pairs complementary to the gene and capable of amplifying the gene.

The kit preferably detects the marine ecosystem response corresponding to polycyclic aromatic hydrocarbon exposure of the genotoxic inducement level of pink sea cockscomb, but is not limited thereto.

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

The kit may further comprise a pink sea cockscomb.

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 wash buffer solution. The present invention is not limited thereto and may include any reaction reagents required for RT-PCR reactions known to those skilled in the art.

The inventors have found that by identifying 32 gene groups whose expression increases or decreases in response to polycyclic aromatic hydrocarbon exposure, the genes can be used to diagnose the state of the marine ecosystem following polycyclic aromatic hydrocarbon exposure. Therefore, the gene corresponding to the polycyclic aromatic hydrocarbon exposure of the present invention can be usefully used as a kit for stress detection and health diagnosis of marine ecosystems including primer pairs thereof.

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 to the following examples.

Pink Sea Cockscomb  Culture and Polycyclic Aromatic Hydrocarbons  exposure

<1-1> Pink sea cockscomb  culture

Scleronephthya collected from the coast of Seogwipo gracillimum ) was incubated in natural seawater through three types of filters (100, 10 and 1 μm). The water temperature was fixed at 22 ° C., the annual average water temperature of Seogwipo area using an underwater heater, and was incubated for more than one week. The photoperiod was adjusted to 14:10.

<1-2> Pink sea cockscomb Polycyclic Aromatic Hydrocarbons  exposure

The pink sea mandrami cultured as in Example <1-1> was exposed to 100 ppb polycyclic aromatic hydrocarbon standard sample (Supelco, USA) and incubated for 24 hours, and the control group not treated with polycyclic aromatic hydrocarbon was treated at 22 ° C. Incubated for 24 hours. The official name of the polycyclic aromatic hydrocarbon is PAH Mix 525, acenaphthylene, fluorene, phenanthrene, anthracene, pyrene, benzo (a) anthracene (Benzo (a) ) anthracene, chrysene, benzo (b) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene (Benzo (a) pyrene), Indeno (1,2,3-cd) pyrene, Dibenzo (a, h) anthracene, Benso (ghi 13 representative polycyclic aromatic hydrocarbons, such as Benxo (ghi) perylene, were mixed at a concentration of 500 μg / ml.

Polycyclic Aromatic Hydrocarbons  By exposure Pink sea cockscomb  Genetic change confirmation

<2-1> RNA Separation of

From the experimental group exposed to the polycyclic aromatic hydrocarbons obtained in Example <1-2> or unexposed pink seawater cockscomb tissue into a powder using a liquid nitrogen in a mortar, and lysis The solution [35 mM EDTA, 0.7 M LiCl, 7% SDS, 200 mM Tris-Cl pH 9.0]] was added to homogenize. The same amount of phenol solution was added and mixed well, then centrifuged for 10 minutes, the supernatant was taken up into a new tube and 1/3 of the total volume of 8 M lithium chloride (LiCl) was added. After mixing well, the mixture was left at 4 ° C. for at least 2 hours. After standing, the supernatant was removed by centrifugation for about 30 minutes, and the precipitate was taken and dissolved in 300 µl of precipitated water. 1/10 volume of 3 M sodium acetate (pH 5.2) and the same amount of isopropanol were added and centrifuged for about 30 minutes to remove the supernatant and the precipitate was taken. 50 μl of 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, the RNA was dissolved in an appropriate amount of DEPC-treated water.

<2-2> cDNA Synthesis of

GeneFishing ^™ DEG kits (Seegene, Korea) were used to isolate specific genes corresponding to polycyclic aromatic hydrocarbon exposure. Total mRNA (3.0 μg) extracted in Example <2-1> was used as a template, and cDNA of the experimental group and the control group was synthesized using dT-ACP1 as a primer and MMLV RT-ase as a reaction enzyme.

<2-3> Isolation and Sequence Analysis of Specific Gene Fragments

Polymerase chain reaction (PCR) was carried out using dT-ACP2 and arbitary ACPs (120 species) as primers as the first strand cDNA synthesized. The PCR condition was 1 minute at 94 ° C; 3 minutes at 50 ° C .; 1 minute at 72 ° C., 40 seconds at 94 ° C .; 40 seconds at 65 ° C .; 40 seconds was performed 40 times at 72 degreeC, and it carried out for 5 minutes at 72 degreeC. PCR products were separated using a 2% agarose gel, and the specifically amplified PCR products identified through the purification were cloned using a T-vector system, and the plasmids were extracted to extract bases. The sequence was analyzed.

As a result, as shown in Figure 1, compared with the control group was confirmed 32 genes increased or decreased expression by polycyclic aromatic hydrocarbon exposure, of which 18 genes increased expression and 14 genes decreased expression . In addition, each nucleic acid sequence described in SEQ ID NOS: 1 to 32 was identified through analysis of the gene sequence.

As described above, the gene of the pink sea cockscomb corresponding to the polycyclic aromatic hydrocarbon exposure of the present invention is a gene specific for polycyclic aromatic hydrocarbon exposure, and a biosensor for monitoring and diagnosing the pollution degree of persistent organic toxic substances and the marine ecosystem status accordingly. It can be usefully used as a method, and can be effectively used for stress detection and health diagnosis methods of marine ecosystems.

Attach an electronic file to a sequence list

Claims (12)

Micro for confirming exposure to polycyclic aromatic hydrocarbons of genotoxicity-induced levels in which oligonucleotides or their complementary strand molecules, all or fragments of the nucleic acid sequence of gene (calmodulin) described in SEQ ID NO: 1, are integrated Array chip.
The microarray chip according to claim 1, wherein the gene is derived from Scleronephthya gracillimum .
The microarray chip according to claim 1, wherein the microarray chip further comprises oligonucleotides or complementary strand molecules thereof which are all or fragments of nucleic acid sequences of any one or more genes selected from the group:
Gene described by SEQ ID NO: 2 (cytosolic malate dehydrogenase), gene described by SEQ ID NO: 3 (40S ribosomal protein S23), gene described by SEQ ID NO: 4 (protein disulfide isomerase), gene described by SEQ ID NO: 5 (dipeptidyl aminopeptidase IV), the gene described in SEQ ID NO: 6 (proteasome beta 3 subunit), the gene described in SEQ ID NO: 7 (ATP synthase F0 subunit 6), the gene described in SEQ ID NO: 8 (40S ribosomal protein S8), SEQ ID NO: 9 Gene described (myosin heavy chain), gene described by SEQ ID NO: 10 (MAK10 homolog, amino-acid N-acetyltransferase subunit), gene described by SEQ ID NO: 11 (heletron 5 helitron-like transposon replicase / helicase / endonuclease), The gene described in SEQ ID NO: 12 (ral guanine nucleotide dissociation stimulator-like 1), the gene described in SEQ ID NO: 13 (allene oxide synthase / 8R-lipoxygenase fusion protein), described in SEQ ID NO: 14 Is a gene (triacylglycerol lipase-like protein), a gene described in SEQ ID NO: 15 (bromodomain adjacent to zinc finger domain 1A), a gene described in SEQ ID NO: 16 (60S ribosomal protein L6), a gene described in SEQ ID NO: 17 (short -chain dehydrogenase / reductase 9), gene described by SEQ ID NO: 18 (cardiac troponin C), gene described by SEQ ID NO: 19 (calcium dependent mitochondrial carrier protein), gene described by SEQ ID NO: 20 (mannose-binding lectin 2) , Gene described by SEQ ID NO: 21 {CXXC finger 1 (PHD domain), isoform}, gene described by SEQ ID NO: 22 (myosin light chain kinase isoform 3A), gene described by SEQ ID NO: 23 (transcriptional regulator, XRE family) , A gene described in SEQ ID NO: 24 (ubiquitin-ptotein ligase), a gene described in SEQ ID NO: 25 (astacin protease 4), a gene described in SEQ ID NO: 26 {poly (ADP-ribose) polymerase 4}, to SEQ ID NO: 27 Gene (ADAM metallopeptidase domain 28 (ADAM28)), gene described by SEQ ID NO: 28 (Collagen, type VII), gene described by SEQ ID NO: 29 (eukaryotic translation initiation factor 4 gamma), gene described by SEQ ID NO: 30 (peptidylglycine alpha-hydroxylating monooxygenase), a gene described in SEQ ID NO: 31 (Ras-related and estrogen-regulated growth inhibitor) and a gene described in SEQ ID NO: 32 (small acid-soluble spore protein).
The method of claim 3, wherein the gene described in SEQ ID NO: 1 (calmodulin), the gene described in SEQ ID NO: 2 (cytosolic malate dehydrogenase), the gene described in SEQ ID NO: 3 (40S ribosomal protein S23), described in SEQ ID NO: 4 Gene (protein disulfide isomerase), gene described by SEQ ID NO: 5 (dipeptidyl aminopeptidase IV), gene described by SEQ ID NO: 6 (proteasome beta 3 subunit), gene described by SEQ ID NO: 7 (ATP synthase F0 subunit 6), sequence The gene described in SEQ ID NO: 8 (40S ribosomal protein S8), the gene described in SEQ ID NO: 9 (myosin heavy chain), the gene described in SEQ ID NO: 10 (MAK10 homolog, amino-acid N-acetyltransferase subunit), SEQ ID NO: 11 Gene described (heletron 5 helitron-like transposon replicase / helicase / endonuclease), gene described by SEQ ID NO: 12 (ral guanine nucleotide dissociation stimulator-like 1), gene described by SEQ ID NO: 13 oxide synthase / 8R-lipoxygenase fusion protein), gene represented by SEQ ID NO: 14 (triacylglycerol lipase-like protein), gene described by SEQ ID NO: 15 (bromodomain adjacent to zinc finger domain 1A), gene described by SEQ ID NO: 16 60S ribosomal protein L6), the gene described by SEQ ID NO: 17 (short-chain dehydrogenase / reductase 9) and the gene described by SEQ ID NO: 18 (cardiac troponin C) have increased expression in response to polycyclic aromatic hydrocarbon exposure, Calcium dependent mitochondrial carrier protein (19), gene (mannose-binding lectin 2) (SEQ ID NO: 20), gene (CXXC finger 1 (PHD domain), isoform), SEQ ID NO: 22 Genes described in (myosin light chain kinase isoform 3A), genes described in SEQ ID NO: 23 (transcriptional regulator, XRE family), genes described in SEQ ID NO: 24 (ubiquitin-ptotein ligase), A gene described by SEQ ID NO: 25 (astacin protease 4), a gene described by SEQ ID NO: 26 {poly (ADP-ribose) polymerase 4}, a gene described by SEQ ID NO: 27 (ADAM metallopeptidase domain 28 (ADAM28)}, SEQ ID NO: 28 Gene (Collagen, type VII), gene described in SEQ ID NO: 29 (eukaryotic translation initiation factor 4 gamma), gene described in SEQ ID NO: 30 (peptidylglycine alpha-hydroxylating monooxygenase), gene described in SEQ ID NO: 31 Ras-related and estrogen-regulated growth inhibitor) and the gene (small acid-soluble spore protein) described in SEQ ID NO: 32 is a microarray chip characterized in that the expression is reduced in response to polycyclic aromatic hydrocarbon exposure.
1) separating the RNA from the pink sea cockscomb of the experimental group treated with seawater to confirm exposure to polycyclic aromatic hydrocarbons, and the pink sea cockscomb of the control group treated with seawater not containing polycyclic aromatic hydrocarbons;
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 of claim 1;
4) analyzing the reacted microarray chip; And
5) A method of confirming exposure to polycyclic aromatic hydrocarbons of genotoxicity induced levels, 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 group.
The method of claim 5, wherein the fluorescent material of step 2) is selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), and rhodamine How to feature.
1) separating the RNA from the pink sea cockscomb of the experimental group treated with seawater to confirm exposure to polycyclic aromatic hydrocarbons, and the pink sea cockscomb of the control group treated with seawater not containing polycyclic aromatic hydrocarbons;
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) Method of confirming the exposure to polycyclic aromatic hydrocarbons of the genotoxic induction level comprising the step of confirming the expression level by comparing the gene product of step 2) with the control.
Claim 1 kit comprising a microarray chip, genotoxic induction level of exposure to polycyclic aromatic hydrocarbons (Polycyclic aromatic hydrocarbons) kit.
The method of claim 8, further comprising any one selected from the group of fluorescent substances consisting of streptadin-like phosphatease conjugate, chemilurorensce and chemiluminescent. Kit characterized in that.
The method of claim 8, 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. Kit comprising any one further.
The kit for confirming exposure to polycyclic aromatic hydrocarbons of genotoxicity-induced levels, comprising primer pairs complementary to the genes integrated in the microarray chip of claim 1 and amplifying the genes.
The method of claim 11, further comprising at least one selected from the group of reverse reagents for synthesizing cDNA from RNA, cNTPs and rNTPs (premixed or separated feed), DNA polymerase, and a reaction buffer group. Kit characterized in that.

Images