KR20110011016A - Seawater temperature change responsive genes in scleronephthya gracillimum and the method for diagnosing marine ecosystem using the same - Google Patents

Abstract

PURPOSE: A gene of Scleronephthya gracillimum which responds to water temperature change and a method for diagnosing marine ecosystem using the same are provided to check stress on marine ecosystem. CONSTITUTION: A microarray chip for diagnosing stress of marine ecosystem comprises: entire nucleic acid sequence of Scleronephthya gracillimum-derived gene, oligonucleotide which is a fragment thereof or complementary strand molecule thereof. A method for diagnosing stress of marine ecosystem comprises: a step of isolating each RNA from Scleronephthya gracillimum of an experimental group and Scleronephthya gracillimum as a control group; a step of synthesizing the RNAs into cDNA and labeling with different fluorescence; a step of hybridizing the cDNA on a microarray chip; a step of analyzing the reacted microarray chip; and a step of comparing the expression level with the control group.

Description

Seawater temperature change responsive genes in Scleronephthya gracillimum and the method for diagnosing marine ecosystem using the same}

The present invention relates to a gene of pink sea cockscomb corresponding to changes in sea water temperature and a marine ecosystem 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, they are a major contributor to our country's marine biodiversity. Therefore, in order to preserve marine biodiversity on the coast of Jeju from climate change, the protection of this species should be a priority.

Climate change refers to the change of the Earth's climate that is changing in various time and space. It refers to the gradual change of the current climate system by natural and artificial factors. In addition to the internal factors of the atmosphere, oceans, land, snow, and the biosphere itself, there are external factors such as increased aerosols (floating particulates) in the eruption of volcanic eruptions, changes in solar activity, and the astronomical relative position of the sun and the earth. Anthropogenic factors include changes in the composition of the atmosphere such as carbon dioxide, land cover changes, and deforestation due to overuse of fossil fuels. As a result of climate change, various environmental problems are deteriorating, including abnormal weather, global warming, and rising sea temperature.

In particular, in recent years, global warming is accelerating due to an increase in atmospheric concentration of greenhouse gases caused by human activities. Due to the greenhouse effect, the global surface temperature, an indicator of global warming, has risen by 0.74 ± 0.18 ° C over the last 100 years (1906 ~ 2005). Due to global warming, sea temperatures are rising, and glaciers in the world except for the polar regions are decreasing. Such rapid changes in water temperature have a major impact on marine ecosystems. That is, it alters the metabolism of marine organisms, resulting in disturbance of marine fisheries and marine fauna and flora.

In order to respond to such environmental changes, marine organisms actively cope with environmental changes by controlling the physiology or metabolism by controlling the expression level of their specific genes. Therefore, by identifying genes whose expression levels change due to external environmental changes such as climate change, and using them as biomarkers, if the gene expression changes of organisms responding to environmental changes are used, not only the information on the environmental changes in a specific region, but also these environmental changes Can provide information on the effects of phenomena on life and health diagnosis of the organism. These techniques can detect even minor changes in the environment, have early diagnostic value, and have future predictive capabilities through consideration of gene function. In the present situation where there is a risk of disturbance of ecosystem caused by climate change, the necessity of developing advanced technology that accurately diagnoses the impact of environmental change and the ecosystem ripple effect through analysis of genes and nucleic acid materials, which are the basics of life phenomenon, is urgently needed. Do.

Accordingly, the inventors of the present invention, while discovering biomarkers corresponding to changes in sea temperature, are known as a pink sea mandrel ( Scleronephthya) , which is a kind of soft coral that plays a large role in maintaining marine biodiversity. gracillimum ), and the change of expression level of specific genes by temperature change was identified, and suggesting that temperature-specific gene candidate group could be useful as a biosensor to grasp the degree of seawater temperature change and the status of marine ecosystem accordingly. The present invention has been completed.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for detecting stress and health diagnosis of a pink sea cockscomb gene corresponding to seawater temperature change due to climate change and marine ecosystem using the same.

In order to achieve the above object, the present invention provides an oligonucleotide comprising all or part of a gene sequence whose expression increases or decreases in response to changes in sea temperature, or an marine ecosystem in which oligonucleotide molecules complementary to the oligonucleotide are integrated. To provide a microarray chip for stress detection and medical examination.

The present invention also provides a stress detection and health diagnosis method of the marine ecosystem using the gene.

In addition, the present invention provides a kit for stress detection and health diagnostics 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 gene of the pink sea cockscomb corresponding to the seawater temperature change of the present invention and the marine ecosystem diagnosis method using the same, by measuring the change in the expression amount of the specific gene of the pink sea cockscomb corresponding to the seawater temperature change according to the climate change, In addition, it can be usefully used as a biosensor and a diagnostic method for diagnosing stress and health of marine ecosystems.

Hereinafter, the present invention will be described in detail.

The present invention provides genes for stress detection and health diagnosis of marine ecosystems corresponding to changes in sea water temperature consisting of the following groups:

The gene described by SEQ ID NO: 1 (protein disulfide isomerase), the gene described by SEQ ID NO: 2 (polyglutamine binding protein variant 4), the gene described by SEQ ID NO: 3 (calmodulin), the gene described by SEQ ID NO: 4 (calreticulin), Ferritin protein as shown in SEQ ID NO: 5, gene as described in SEQ ID NO: 6 (small GTPase), gene as described in SEQ ID NO: 7 (40S ribosomal protein S23), gene as described as SEQ ID NO: 8 (mannose-binding lectin 2), the gene described in SEQ ID NO: 9 (eukaryotic translation initiation factor 4), the gene described in SEQ ID NO: 10 (proteasome beta 3 subunit), the gene described in SEQ ID NO: 11 (ATP systhase subunit 8), SEQ ID NO: 12 Gene described (ribosomal protein S8), gene described as SEQ ID NO: 13 (myosin heavy chain), gene described as SEQ ID NO: 14 (heletron 5 helitron-like transposon replicase / helicase / endonuclease), sequence Triacylglycerol lipase-like protein as described in No. 15, gene as described in SEQ ID NO: 16 (bromodomain adjacent to zinc finger domain), gene as described in SEQ ID NO: 17 (ribosomal protein L6), gene as shown in SEQ ID NO: 18 (ADAM metallopeptidase domain 19), the gene described in SEQ ID NO: 19 (calcium dependent mitochondrial carrier protein), the gene described in SEQ ID NO: 20 (PAX interacting protein 1-reverse), the gene described in SEQ ID NO: 21 [poly (ADP- ribose) polymerase 4], the gene described in SEQ ID NO: 22 (hydroxysteroid (17-beta) dehydrogenase), the gene described in SEQ ID NO: 23 (collagen, type XXV), the gene described in SEQ ID NO: 24 (peptidylglycine alpha-hydroxylating monooxygenase And ras-related and estrogen-regulated growth inhibitors.

In the gene group, a gene whose expression is increased in response to a temperature increase preferably includes the following genes, but is not limited thereto.

The gene described by SEQ ID NO: 1 (protein disulfide isomerase), the gene described by SEQ ID NO: 2 (polyglutamine binding protein variant 4), the gene described by SEQ ID NO: 3 (calmodulin), the gene described by SEQ ID NO: 4 (calreticulin), Ferritin protein as shown in SEQ ID NO: 5, gene as described in SEQ ID NO: 6 (small GTPase), gene as described in SEQ ID NO: 7 (40S ribosomal protein S23), gene as described as SEQ ID NO: 8 (mannose-binding lectin 2), eukaryotic translation initiation factor 4, SEQ ID NO: 10 gene (proteasome beta 3 subunit), gene described in SEQ ID NO: 11 (ATP systhase subunit 8), SEQ ID NO: 12 Gene described in (ribosomal protein S8), gene described in SEQ ID NO: 13 (myosin heavy chain), gene described in SEQ ID NO: 14 (heletron 5 helitron-like transposon replicase / helicase / endonuclease), sequence Triacylglycerol lipase-like protein, genes described in SEQ ID NO: 16 (bromodomain adjacent to zinc finger domain), genes described in SEQ ID NO: 17 (ribosomal protein L6), and genes described in SEQ ID NO: 18 (ADAM metallopeptidase domain 19).

In the gene group, a gene whose expression decreases in response to an increase in temperature preferably includes the following genes, but is not limited thereto.

Calcium dependent mitochondrial carrier protein (SEQ ID NO: 19), PAX interacting protein 1-reverse (SEQ ID NO: 20), gene (SEQ ID NO: 21) [poly (ADP-ribose) polymerase 4], sequence The gene described in SEQ ID NO: 22 (hydroxysteroid (17-beta) dehydrogenase), the gene described in SEQ ID NO: 23 (collagen, type XXV), the gene described in SEQ ID NO: 24 (peptidylglycine alpha-hydroxylating monooxygenase), and SEQ ID NO: 25 Ras-related and estrogen-regulated growth inhibitors.

The present inventors investigated the genes that change the expression level by synthesizing the pink sea cockscomb cDNA after culturing the pink sea cockscomb at high temperature (28 ℃) in order to discover the pink sea cockscomb gene corresponding to the sea water temperature change. Specifically, mRNA was isolated from pink seawater tissues cultured at 22 ° C. (control) or 28 ° C. (experimental), and cDNA was synthesized using the purified mRNA using GeneFishing ^™ 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, 25 genes whose expression increased or decreased due to temperature increase were obtained. Climate change causes changes in sea temperature, and marine life causes changes in physiology and metabolism in response to these changes. Therefore, by confirming the change in the amount of gene expression of marine organisms can be used as a biomarker that can determine the stress and health status of marine organisms according to the external environment changes. Therefore, since the genes have a change in the expression of the genes in response to the temperature rise carried out in the present invention, it can be seen that they can function as genes for diagnosing stress and detecting the state of the marine ecosystem according to changes in seawater temperature.

In addition, the present invention is a stress of marine ecosystem in which cDNA fragments, oligonucleotides, gene fragments complementary to the cDNA fragments, or oligonucleotide molecules complementary to the oligonucleotides are integrated. Provided is a microarray chip for detection and medical examination.

The microarray chip preferably detects marine ecosystem responses corresponding to changes in sea temperature due to climate change, but is not limited thereto.

The present inventors have found that by identifying 25 gene groups whose expression increases or decreases in response to changes in sea water temperature, the genes can be used to diagnose the state of marine ecosystems according to sea water temperature changes. Therefore, the gene corresponding to the seawater temperature change of the present invention can be usefully used as a microarray chip for stress detection and health diagnosis.

In addition, the present invention provides a stress detection and health diagnostic method of the marine ecosystem using the gene of the present invention.

The present invention

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) provides a method for detecting stress and health diagnosis of the marine ecosystem comprising the step of confirming the expression level of the gene of the present invention in the analyzed data of step 4) compared to the control.

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.

The present inventors have found that by identifying 25 gene groups whose expression increases or decreases in response to changes in sea water temperature, the genes can be used to diagnose the state of marine ecosystems according to sea water temperature changes. Therefore, the gene corresponding to the seawater temperature change of the present invention can be usefully used for stress detection and diagnosis of marine ecosystem using a microarray chip.

In addition,

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) provides a method for detecting stress and health diagnosis of the marine ecosystem comprising the step of comparing the gene product of step 2) with the control to confirm the expression level.

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

The present inventors have found that by identifying 25 gene groups whose expression increases or decreases in response to changes in sea water temperature, the genes can be used to diagnose the state of marine ecosystems according to sea water temperature changes. Therefore, the gene corresponding to the seawater temperature change of the present invention can be usefully used for stress detection and health diagnosis of marine ecosystem using RT-PCR.

The present invention also provides a kit for stress detection and health diagnosis of marine ecosystems comprising the microarray chip of the present invention.

The kit preferably detects marine ecosystem responses corresponding to changes in sea temperature due to climate change, 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 may include all reaction reagents required for hybridization reactions known to those skilled in the art.

The present inventors have found that by identifying 25 gene groups whose expression increases or decreases in response to changes in sea water temperature, the genes can be used to diagnose the state of marine ecosystems according to sea water temperature changes. Therefore, the gene corresponding to the seawater temperature change 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 diagnosis of marine ecosystems comprising primer pairs complementary to the gene of the present invention and capable of amplifying the gene.

The kid is preferably to detect a marine ecosystem response corresponding to changes in sea temperature due to climate change, 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 present inventors have found that by identifying 25 gene groups whose expression increases or decreases in response to changes in sea water temperature, the genes can be used to diagnose the state of marine ecosystems according to sea water temperature changes. Therefore, the gene corresponding to the seawater temperature change of the present invention can be usefully used as a stress detection and health diagnostic kit of the marine ecosystem including a primer pair.

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.

< Example  1> Pink Sea Cockscomb  Incubation and high temperature exposure

<1-1> Pink sea cockscomb  culture

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

<1-2> Pink sea cockscomb  High temperature exposure

The pink sea cockscomb of Example <1-1> was exposed to 28 ° C., which is 2 ° C. higher than 26 ° C., the highest water temperature of summer in Seogwipo, and incubated for 24 hours, and the control group was incubated at 22 ° C. for 24 hours.

< Example  2> Genetic change measurement by temperature rise

<2-1> RNA Separation of

In the mortar and pesticide, the pink seamandramy tissues of the test group and the unexposed control group obtained in Example <1-2> were powdered using liquid nitrogen in a mortar, and a lysis 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, ethanol solution was removed, and the precipitated RNA was dried. After drying, it was dissolved in an appropriate amount of DEPC-treated water.

<2-2> cDNA  synthesis

GeneFishing ^TM DEG kits (Seegene, Korea) were used to isolate specific genes corresponding to temperature changes. Using the extracted total mRNA (3.0 ㎍) as a template dT- ACP1 as a primer and MMLV RT-ase as the reaction enzyme was synthesized cDNA of the control and experimental groups.

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

 PCR was carried out using dT-ACP2 and arbitary ACPs (120 species) as primers using the synthesized first strand cDNA as a template. 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 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. Sequencing was performed.

As a result, as shown in FIG. 1, 25 genes whose expression was increased or decreased by temperature increase compared to the control group were identified, and the nucleic acid sequences described in SEQ ID NOs: 1 to 25 were identified through sequence analysis.

As described above, the gene of the pink sea cockscomb corresponding to the seawater temperature change of the present invention can be usefully used as a biosensor for monitoring and diagnosing the degree of seawater temperature change and the marine ecosystem status according to the temperature change specific gene, It can be effectively used for stress detection and medical examination of marine ecosystems.

1 is a diagram showing the difference in the gene expression of pink sea cockscomb cultured at 22 ℃ or 28 ℃.

<110> Korea Ocean Research and Development Institute <120> Seawater temperature change responsive genes in Scleronephthya          gracillimum and the method for diagnosing marine ecosystem using          the same. <130> 9P-06-55 <160> 25 <170> KopatentIn 1.71 <210> 1 <211> 448 <212> DNA <213> Scleronephthya gracillimum <400> 1 cgtgactttg gctccgaagt acgaagaggc tgcaaggatg attgcaaaag gacacttaaa 60 agataaagtt catttagtta agatccaatg cgacagcgag aaaggaagag aaatatgttc 120 aacaaataaa gtagaaggat ttccgactat caatgtgtat caaaaaggaa agaaacttga 180 acaatacgta ggcgctagaa caccagatgc tttcgttaag tacgtggagg aagttgtaaa 240 gacaggaaag cctcaattta ctggaaaaca aaggaacgac caacaagtct gcctggccaa 300 ggatttcaga aaatatgtta cccaaaaatc gtcatgaagg aaatatagtt agacgttaaa 360 ttttgaacga tgttacgtat ttacgtgtac atatcaagag tataaatatt tttgaaatgt 420 aattattaaa tcaaaggatt ttcgtgaa 448 <210> 2 <211> 502 <212> DNA <213> Scleronephthya gracillimum <400> 2 aaaaaatttt tattttatat ctactttaca acttagtaaa atgggtttgg tcgtttggca 60 acaaattttg atttgaactg ggatttcatc acccgatgtg gcccccccat gaagcgaatg 120 cctggtaaac aatcactagt gaattcgcgg ccgcctgcag gtcgaccata tgggaaagct 180 cccaacgcgt tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta 240 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 300 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 360 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 420 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 480 gctcactgac tcgctgcgct cg 502 <210> 3 <211> 430 <212> DNA <213> Scleronephthya gracillimum <400> 3 aattacgtca tgtgatgaca aatcttggtg aaaaattgac agatgaagag gttgatgaga 60 tgatcagaga agctgatatt gatggagatg gacaagttaa ctatgaagaa tttgtgaaaa 120 tgatgacttc caagtagatg tttcaatttt acgacaagtt ctggttatga catggccacg 180 aataatttct gatggctgac ttagctcctg cctcccatat taaataactt ttatttcact 240 tttcatttaa ttgtattttt atttaatgta agcactaggt taggaataag aatactattc 300 aagcaatcta ttagttgaat atttatacgt aagtagatat tgttgcatgg tgatatagat 360 gtaaattata cttaatgtaa tttcctgtct tagtttccaa ccacaatgta attaaatacc 420 cattttatta 430 <210> 4 <211> 765 <212> DNA <213> Scleronephthya gracillimum <400> 4 ctttgtaata agttgatggt aaaaatgggt cttttaaggc tatttgcttg tgttcttttt 60 gttttctcaa ttgtggaatc aacagtgtat tttaaggaaa cctttggtga tgcggactgg 120 gagaaacggt gggttagctc gaccaagaag ggcagtgatg ctggaaagtt tgttttatct 180 gctggcaagt tttataatga cccggagcta gataaaggaa ttcagacgag tcaagatgct 240 aaattttacg gtctctcagc gaaatttgat gaagcattct caaacaagga caaagatctt 300 gtgattcaat tcacagttaa acatgagcaa acgattgact gtgggggtgg atacgtcaag 360 gtatttggaa gtgacttaga ccaagctggt atgcatggag actcaccata gaacatcatg 420 tttggacctg atatctgtgg acctggaaca aagaaagttc atgtaatctt caactacaag 480 ggaaagaacc ttttgaccaa gaaagatatt agatgcaagg atgatgaaat gacccatctt 540 tatacattga ttgtgaaacc agacaatact tatgaagtaa gaatagacaa tgaaaaagtt 600 gaaagtggtg aactagagca agattgggaa tttttgcctg agaagaagat aaaagatcct 660 gaagctaaga agcctgatga ttgggtggat gatgctaaaa tccctgatcc cgaagacaag 720 aaacctgatg actatgacaa accacaacat attcctgacc ctgat 765 <210> 5 <211> 513 <212> DNA <213> Scleronephthya gracillimum <400> 5 acgttgcatt ggctggattt cacaaatact tcaagaaagc ctcagatgag gaacgagaac 60 acgctgaaaa actgatgaag tttcagaatc gtcgtggtgg acgtattgtt ttgaaggaca 120 taaagaaacc tgagaaagat gaatggggca atggactgga tgctatgaag gttgcactgg 180 cattggaaaa gaccgtgaat gaagcattgt tccagttaca tgaagttgct gtcaccaaca 240 aggatgatga gatggctgat tttattgaag ccaactactt gcatgagcaa actgatgcca 300 tcaaggaatt gtcagatcat attaccaacc ttgaacgtgt tggtgaaggc ttgggtgaat 360 accaatttga caagctcaca ttgaatggtg atgactaatt tcttacttgt tgatgaactg 420 aattcatgta accaggataa tcaattagct tagtaacaat actgtaatga tcaagtaaag 480 gaaaaaggga ttaaaattat taaagcaaaa aaa 513 <210> 6 <211> 235 <212> DNA <213> Scleronephthya gracillimum <400> 6 tgattagaag ggcaaactgt gtacaattgc gtaactagcg ttaaatttat tcactgggtg 60 ggctgttgtg tgtatataga catattattg tgtacaaaga cagtcttatt agacgaaatt 120 gtaaataaag taattagaaa ccgaatggaa tatttaatgt ggatagaaac ttctttatcg 180 agtgatattg taaaaaggaa atatttgaat attaaattgt gcatattaac tataa 235 <210> 7 <211> 322 <212> DNA <213> Scleronephthya gracillimum <400> 7 catgcgaagg gaatcgtact cgagaaagta ggtgtcgagg ccaaacagcc taactcggcc 60 atccgtaagt gtgtcagggt ccagctcatc aagaacggaa agaaaatcac cgccttcgtg 120 ccccgtgatg gttgcctcaa ctacattgaa gagaacgacg aggtgttggt agccgggttc 180 ggtaggaagg gtcacgccgt gggagatatc cccggagtac gattcaagat cgtcaaggtc 240 gccaacgtct ccctactcgc cctgtacaag gagaagaagg agaggcctcg atcataaaca 300 ttctctacag ttatatacaa ta 322 <210> 8 <211> 334 <212> DNA <213> Scleronephthya gracillimum <400> 8 cgtttgcacg ttctaaaggc tcgcgacaaa cctgggttgg cgtttttagg cgatatgaca 60 aacgttttat caacgttgaa gggagagacc aaacctacac caactggaac cgaggagaac 120 caaacaacag cggcggtaac gagaactgtg ttgtcatgta taccaacact gctagatgga 180 atgacgcaag ttgtactact cgccatgatt tcgtctgtga aatcaaactt tgattgtgtg 240 cataaatcag ctaagtgagc tttttgtggc ctatacgtct gggcctacaa cgtatgttag 300 ctatttgcag taataataaa atcgtgattt aatc 334 <210> 9 <211> 195 <212> DNA <213> Scleronephthya gracillimum <400> 9 tgttcagcgt tgcttatgca aatatgtgcc gatgtctatt cacgataaaa gtttccgatg 60 tggatggaaa agaagtgtct ttccgaaagt tgctgctgaa tagatgtcag aaagagtttg 120 aaaaagaaaa ggagtgcgaa aatgcgttat tagaaaagaa tactcaactt gaaggactat 180 cggaggagga aatga 195 <210> 10 <211> 337 <212> DNA <213> Scleronephthya gracillimum <400> 10 tgcagaagat tttgttgtta gcggcacctg tactgagcag atgtatggta tgtgcgagtc 60 gctatggcaa cctgatttgg cgccggacga tttgttcgag accatctcac aagcactcat 120 gaacgcagtc gaccgtgatg ctgtcagtgg ttggggtgct gtggttcata ttgtcgaaaa 180 agataagatc acgactcgcc atttaaagac aagaatggac tagtactaat atgcaaaaaa 240 gtgataaaca atgttctgaa gtacttctgt aaaattctat gaacacatag tgaactctta 300 ctaataaatg ttaaaaatcg ttttcaatgt aaaaaaa 337 <210> 11 <211> 547 <212> DNA <213> Scleronephthya gracillimum <400> 11 ggagccctac caacaatctc agcaatccta ttaagcctta tatcattaaa tatattagga 60 ctattacctt atacaatttc aataacatcc cacttaatga caactctatc attagggcta 120 ccaatctgag gctcaataat tctcatctca ttagcaatta gcaggaaaac cactagggcg 180 catttactcc ccccaaacac cccttcattt attgccaggt tcctgttact tgtagaagcc 240 ctaagatctt taattcgccc cctgaccctc tctttccgtt tagcagctaa catcagagct 300 ggacacgtaa tcctaggaat aatatcatct ggtgccactt atctcatata tacacaccca 360 acagctgcta ttctaccaat aatagtaaga agcttttatt ttgtattcga gatcgcaatt 420 tgcgcagtac aagcatttgt atttgttctt cttattgcta tgtactctaa tgattacatt 480 agataacaca ataagcttaa attttaataa ttattccaaa ttacatactc caattatata 540 accttat 547 <210> 12 <211> 135 <212> DNA <213> Scleronephthya gracillimum <400> 12 ggccagagtg gtcgatgtga tggttatatc ttggaaggca aagaacttga gttctacgtc 60 aagaaaatta gggcaaagaa gagcaaatag attgtaaaaa tgctgttcaa aacaaaataa 120 acaatttgaa aacaa 135 <210> 13 <211> 255 <212> DNA <213> Scleronephthya gracillimum <400> 13 gcgacgacgc taccgcaaga tgtgcgacca gagaattggt atcgctgtta tccggaggaa 60 tgtgaggaag tatttgttct tgaagaactg ggcttggtgg aaactttata ccaaggtgaa 120 accattgttg aacgtcgcgc gaactgaaga agaaatgaaa cagaaagaag aggagttggc 180 aaaacttaaa gatgatttag caaaggagaa agaactgaga caatcacttg agaatgagaa 240 gacggaattg attca 255 <210> 14 <211> 211 <212> DNA <213> Scleronephthya gracillimum <400> 14 gactggtttt atagagttga gtatcaacaa agagattcgc ctcatattca tatgttaata 60 tggtagaaga tgctccagtt tttggtgtta ataatgacat acaagtgaca gcatttattg 120 ataaaataat tagctgtaaa aaaccatggg ataatcctca gttacttaaa cttgttaaca 180 ggcaagtgca tcgccattcc ctcacttgct a 211 <210> 15 <211> 529 <212> DNA <213> Scleronephthya gracillimum <400> 15 gaaagcacgg tcattgcata tgacagacaa acacccaaga acaacttgtt tctatacacc 60 tttggccagc caagagtcgg tgattaccaa tacgccttac aacatgatag actggtaccc 120 ataagcttta gggttaccca ctatcgagat cccgtggttc acctgcccac ctgcaaaact 180 ctcttgcctg gaacaccctg tatcgcatac accgggggac cctaccacca tggcaaggag 240 atttactatg gcaacaccgt gatgacgaaa acatcgtctt atagaaagtg tcagggattg 300 ccacacaacg aagacctgaa atgcagtaac aaccctaagg tatgggtaaa gtgcttcacc 360 aacataaaga gctgtatcaa tgatcatcgg cagtattttg gagttcgtgt tggagtctgg 420 tggcgaggtt agagacacga agaagctaag aacacccaat atccttatat aaagagtgga 480 attagacatt taatctgcat gctttttctt gtgcaacgtt atagaacta 529 <210> 16 <211> 315 <212> DNA <213> Scleronephthya gracillimum <400> 16 acaacccatc aaaagaggtg tccttaccgt agttcattat acccgacata gccgcttgga 60 taatttatgt gatgaagttt tcaactatat acgagatcga taccaagaag gcgaagaagt 120 tgatgtaaaa tataaaggag agaagttggt tggctttatc cacaatgtta ttgaacctga 180 aattctaaca aatgggtttg actcccccaa atcaaagaat aaactgaatt caccatctgg 240 taaatctgga aaatctcaag aagttattgt cttgggcaat gagaaaaatg aaagaacaga 300 agtacagagc ccaaa 315 <210> 17 <211> 462 <212> DNA <213> Scleronephthya gracillimum <400> 17 gtggtcattc tcttggctgg aagacaccag ggcaaaagag ttatcttctt gaaacaattg 60 cctggtggtt tgctccttgt cactggcccg tacaaaatca acggtgtacc cctccgtcgt 120 gttccccagt cctacgtgat tgccacacag accaagattg acatcagtgg tgtagacctt 180 ccagaacgtc tcgatgacga ttacttcaaa cgagagaaac ggcaaagaaa acgaactgag 240 gaaatgtttg aggaagagaa agaggaggtc actgttagtg atgaaaggaa ggaagatcaa 300 aaagccgtcg atgaacaact tgctcctttg atatcaaatg aagcacattt gaagaattac 360 ctcaagactt tgttcagttt gagaaaggga caatatcctc atgaaatgat cttctaaatt 420 tgtacaagat taagctacta ataaaatctt gccaaccata aa 462 <210> 18 <211> 559 <212> DNA <213> Scleronephthya gracillimum <400> 18 tactgatcta aagacggtga atggtttatc ctgcaaaaat ggacagggat attgttataa 60 aggggaatgt acaacgtata atgagcaatg taactatttg tggtttggcg gtggttttaa 120 agcgaatgat gtttgttacc aaaggtataa tctacgtggc gatattcatg gttactgcaa 180 aagatattca gcaacaagtt acaaggcttg tacaccagaa aatgttaaat gtggcaaatt 240 atggtgtgtt ggtaacggcc acttagaagc aaaaaatttt ggctttggta ttaaatggag 300 tcgtggtact ataaatggtc acacttgtcg ctctgctgtg atcaacatga atccaggtaa 360 accggcattg ggcactgtct tagatgggac aaaatgtggg agcggcaagg tttgtcaaga 420 caataagtgt gtgtcacttt cagcggctta tggtgggcag ccaaaatgca caaataactg 480 taacggacat ggggtctgta atgagaaggg caactgccat tgtaatccag gatggaaatg 540 tcctgattgt tccaaatcg 559 <210> 19 <211> 386 <212> DNA <213> Scleronephthya gracillimum <400> 19 gaatttgaga agtatgacac caacggcgat ggtaaaatca gcgaagaaga gtttaagaag 60 accctgtctc tgtcagataa ggatgcggcc gaccttttca aaagcttaga taaagatggt 120 aatggcgtcg attgcaatga gttgacaaaa agcaacttgg agttcagatg taaaacaaag 180 gcatgccctg ataacaagaa tggaaaatcc caagatgagt ttttggaaga tgagtgaaat 240 gaacagccct tcctttgttt caaccgtacc tcatttgaca tctaacgatt agtaatgcat 300 gcaatttagg attaactttt tgtcataatc ataatgaact acagtagcta ctttaatact 360 tttcaataaa tccttcttaa ggcaaa 386 <210> 20 <211> 160 <212> DNA <213> Scleronephthya gracillimum <400> 20 caatccacaa catccttacc cctcccatcc tgcacaccaa catcctatac attccaatcc 60 tctcataaaa cctcccccac ctcattccca aacctcattc ctaaacctca ttcccaaact 120 ttatcgggtt ctaagcgtta gaaaccccca ggatgaaaaa 160 <210> 21 <211> 306 <212> DNA <213> Scleronephthya gracillimum <400> 21 attcagtttt tcctgctggt attggctcac acaaaatgat ttctaacaat atatccagtc 60 aaatattgaa caaagatagt ctatcgccct ctgtgaaatc gcttgttcat agcatttggt 120 ccgacgcgat tggcgagcta agttcgttac ttagcgaacc ggttgaacgg ctgaaactgg 180 aaaacattgc taaagctgaa ggaatattac aagctattcg cgaaattttg gacaaaaacg 240 agccttctga tgaaaacttg aggaaatatt ccgaggaatt ttactctctc gttccacaca 300 acgaaa 306 <210> 22 <211> 575 <212> DNA <213> Scleronephthya gracillimum <400> 22 gatgatgtct ggaaagactg acgtgaaaac tacatgcctt tgtccgtaca tggtcaaaac 60 cccattgatt gggaagactt ctgcaaggct tccgtcaatg ttcccactgt tggagcccga 120 ctttgtggca aaagaattgg tggacggcat gctacggaac aagaacatgg tcatcctgcc 180 aaaaataatg accttgcatt tggcgttacc aatgttcatg ccagaaaacg gattgagaat 240 gctataccag ttttatcgca taacagttga ggaggaaacg gcaacaacca tgtttcaacg 300 gcgagaacaa gctgctgcag tcgaggaaaa gaagtcggaa taagaatgca caaaattacg 360 tggcttaaag aaggaataca agaattgtca aaagcgaact ttgaaatgaa ttagttgtta 420 attgcttagg gagaaattga aactttagca tgtcaatatc tttagtgacc gtccttagga 480 ggaacttgaa actttattct ttttaatgtc aatatcttta gctatcatgt attgtgtgta 540 tttattcttt tatggaatat atttttaaat ttgaa 575 <210> 23 <211> 398 <212> DNA <213> Scleronephthya gracillimum <400> 23 gccgggtcta cctggtatcc gtggtataaa aggattcccc ggaatccctg gttgtaaagg 60 tcaaccgggt gagcctggta cagccaatgg cgcggttggt ttagatggac caccagggcc 120 cgaaggtgat cgaggaccaa aaggtccaaa gggtgatccc gtatctggtc ctacgccgga 180 tcaaaaaggt agcaagggat cactgggtga tacaggagcc aaaggaatag agggaccaag 240 aggcagagct ggtccaaaag gtgatgtggg aaaaaagggt atcgatggtg agcagggtct 300 tgaaggtgaa caaggtgata ggggtaaacc gggagattca cccaaacagg tagatgaagt 360 tacagatcat ctgaaaggaa tgcggggaga ggaaggtg 398 <210> 24 <211> 643 <212> DNA <213> Scleronephthya gracillimum <400> 24 catgggtccc gaaatgcacc cgtttgcgtt cagagttcat actcacaagt taggctacgt 60 tgtgactggg tacagaatac gtgatggtaa atggcatctc ataggaaagg gcgacccaag 120 acgacctcag gcattttata aagttgacaa tgaagattcc cttcttcctg gagatacact 180 ggcggctaga tgtacttaca attctatgaa gcgagatcac accacgtata ttggggcgac 240 aggtgcggac gagatgtgca atttctacat gatgtactgg tatgacccgg ccgaaggtca 300 gtccaccgat gcatgtttta attctgaaat acccgatgat gactacccca aggatactaa 360 cgtaccacta cctgcccaga agaaaatgga aatgaaacgg gatatggatg attctggtga 420 aaacgtcttc gattctgaag atgaagaatc atggctttaa atacaaaata tcaagacttc 480 aagattaatt ctacctatac tatatgtact gttaacttat tgctaaatag aaatttaacc 540 actaataata tattgataac tgataatacg agaaacttag cttaacttaa tcatcaagtt 600 cattcataat tttcctgtat gcaaatacac agaagtctat aaa 643 <210> 25 <211> 240 <212> DNA <213> Scleronephthya gracillimum <400> 25 gtatttaacg aaacgtttca tcggagaata ccagagcagt gttgaaacgc acgcctcgac 60 tgaggttgaa attgacgaag aaaaagttgt cgtggaaata tttgatacaa gtgcttcgga 120 agagaagatt ttggagcaca tcaactgggc agacggattc attctggtct tttcaatcac 180 agattactgg agcttatatg aagtagccag actatcgtcc atcatatcgc agacgaaaaa 240                                                                          240  

Claims (13)

Microarray chip for stress detection and health diagnostics of marine ecosystems in which oligonucleotides or complementary strand molecules thereof, which are all or fragments of nucleic acid sequences of any one or more genes selected from the following groups are integrated. The gene described by SEQ ID NO: 1 (protein disulfide isomerase), the gene described by SEQ ID NO: 2 (polyglutamine binding protein variant 4), the gene described by SEQ ID NO: 3 (calmodulin), the gene described by SEQ ID NO: 4 (calreticulin), Ferritin protein as shown in SEQ ID NO: 5, gene as described in SEQ ID NO: 6 (small GTPase), gene as described in SEQ ID NO: 7 (40S ribosomal protein S23), gene as described as SEQ ID NO: 8 (mannose-binding lectin 2), the gene described in SEQ ID NO: 9 (eukaryotic translation initiation factor 4), the gene described in SEQ ID NO: 10 (proteasome beta 3 subunit), the gene described in SEQ ID NO: 11 (ATP systhase subunit 8), SEQ ID NO: 12 Gene described (ribosomal protein S8), gene described by SEQ ID NO: 13 (myosin heavy chain), gene described by SEQ ID NO: 14 (heletron 5 helitron-like transposon replicase / helicase / endonuclease), sequence Triacylglycerol lipase-like protein as described in No. 15, gene as described in SEQ ID NO: 16 (bromodomain adjacent to zinc finger domain), gene as described in SEQ ID NO: 17 (ribosomal protein L6), gene as shown in SEQ ID NO: 18 (ADAM metallopeptidase domain 19), the gene described in SEQ ID NO: 19 (calcium dependent mitochondrial carrier protein), the gene described in SEQ ID NO: 20 (PAX interacting protein 1-reverse), the gene described in SEQ ID NO: 21 [poly (ADP- ribose) polymerase 4], the gene described in SEQ ID NO: 22 (hydroxysteroid (17-beta) dehydrogenase), the gene described in SEQ ID NO: 23 (collagen, type XXV), the gene described in SEQ ID NO: 24 (peptidylglycine alpha-hydroxylating monooxygenase And ras-related and estrogen-regulated growth inhibitors. The microarray chip according to claim 1, wherein the gene is derived from Scleronephthya gracillimum . The microarray chip according to claim 1, wherein the gene is increased or decreased in response to seawater temperature change due to climate change. The microarray chip of claim 1, wherein the microarray chip detects a marine ecosystem response corresponding to a seawater temperature change caused by climate change. 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 cDNA labeled with different fluorescent materials of step 2) with the microarray chip of claim 1; 4) analyzing the reacted microarray chip; And 5) Stress detection and health diagnostic method of the marine ecosystem 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 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 A method for detecting and diagnosing stress in marine ecosystems. 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 (RT-PCR) using a pair of primers complementary to the gene of claim 1 and amplifying the RNA of step 1); And 3) Stress detection and health diagnostic method of the marine ecosystem comprising the step of confirming the expression level by comparing the gene product of step 2) with the control. Claim 1 kit for the stress detection and health diagnostics of the marine ecosystem, characterized in that it comprises a microarray chip. The kit of claim 8, wherein the kit detects a marine ecosystem response corresponding to a change in sea temperature due to climate change. 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 for stress detection and health diagnostics of marine ecosystem, 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 for stress detection and medical examination of the marine ecosystem, further comprising any one. The kit for stress detection and health diagnosis of marine ecosystem comprising a primer pair that is complementary to the gene integrated in the microarray chip of claim 1 and amplifies the gene. The kit according to claim 12, wherein the kit detects a marine ecosystem response corresponding to seawater temperature change due to climate change.

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