KR20120131042A - Dissolved oxygen change responsive genes in Mytilus galloprovincialis and the method for diagnosing marine ecosystem using the same - Google Patents

Abstract

PURPOSE: A dissolved oxygen change-responsive Mytilus galloprovincialis gene and a method for diagnosing marine eco-system using the same are provided to detect marine eco-system stress and to diagnose marine eco-system condition. CONSTITUTION: A microarray chip for detecting marine eco-system stress or diagnosing a marine eco-system condition contains nucleic acid sequence oligonucleotides of a specific gene or complementary molecules thereof. The gene is derived from Mytilus galloprovincialis. The expression of the gene is increased or decreased by dissolved oxygen change. A method for detecting the marine eco-system stress or diagnosing marine eco-system condition comprises: a step of isolating RNAs from stress-exposed Mytilus galloprovincialis as an experimental group and a normal Mytilus galloprovincialis as a control group; a step of synthesizing the RNAs and labeling the cDNAs of the experimental group and control group with different fluorescent materials; a step of hybridizing the cDNA on the microarray chip; a step of analyzing the microarray chip; and a step of comparing gene expression levels between the experimental group and control group.

Description

Dissolved oxygen change responsive genes in Mytilus galloprovincialis and the method for diagnosing marine ecosystem using the same}

The present invention relates to a microarray chip for stress detection or health diagnosis of a marine ecosystem, and a method for detecting stress or health diagnosis of a marine ecosystem using the same.

Mediterranean sea urchin ( Mytilus galloprovincialis ) is native to the Mediterranean of Europe, but their distribution has spread throughout the world by the movement of ships. In Korea, it is easily found all over the coast, so it can be used as a good experimental material for evaluating coastal environment using living organisms. It replaces mussels by taking an advantage in the ecological competition with previous mussels. Although it is less commercially available than mussels, it is used as food, and farming is also important for this species.

Hypoxia (hypoxia) in coastal waters means the dissolved oxygen level below 2-3 mg / L when benthic animals begin to die, and the waters lacking dissolved oxygen amount are called poor oxygen waters. The formation of the empty oxygen zone is mainly due to coastal eutrophication and stratification, which causes a large number of primary producers due to eutrophication, so that a large amount of organic matter is supplied to the lower layer, and the respiratory volume of low-level bacteria decomposing organic matter increases rapidly. As the temperature rises in sea level, a stratified layer (or a water temperature medicine layer) is formed, which limits oxygen supply. This results in the formation of vacant oxygen water masses, which are vacant oxygen water masses, in the coastal bottom layer, and the dissolved oxygen gradually decreases to become anoxic when the state persists for a long time. Strata occur frequently in semi-closed or obstructed seas where seawater circulation is poor. In summer, the water temperature weak layer is formed by the large inflow of fresh water by rainfall, and the density density layer is formed strongly by the salt difference between the surface water and the low water. In addition, the inner bay is more polluted with organic matter than the open sea, and frequent oxygen lumps are generated. These oxygen-free waters, or water-free water masses, occur in thousands of kilometers of coastal waters worldwide, including North America, Europe, and the Pacific.

The formation of an empty oxygen environment affects the respiration and physiology of aquatic organisms, disrupts the balance of ecosystems, changes in the movement and spawning patterns of organisms, reduces habitats, increases sensitivity to predators, increases infection rates to external pathogens, It causes changes in the food chain, resulting in a decrease in the species and population of benthic communities. If this effect persists, it causes massive mortality of living in the region, resulting in biodiversity loss, which leads to a loss of coastal productivity, leading to enormous economic losses. In addition, the formation of oxyhydrogen lumps poses a serious threat to aquatic animal populations and the maintenance of genetic diversity. Therefore, efforts to understand and resolve the effects of oxygen reduction on aquatic ecosystems in various ways are necessary.

In response to changes in the environment, organisms actively cope with environmental changes to some extent by changing the physiology or metabolism by controlling the expression level of their specific genes. Therefore, by identifying genes whose expression levels change due to changes in the external environment such as dissolved oxygen levels, 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, Information on the effects of change on life phenomena and on the health of the organism can be obtained. These techniques can detect even minute 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 danger of disturbance of the ecosystem by the formation of the empty oxygen zone, the necessity of the development of advanced technology that accurately diagnoses the impact of environmental change and the ecosystem ripple effect through the analysis of the genes and nucleic acid substances 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 the marine environment, change the expression level of specific genes in the empty oxygen state by reducing the dissolved oxygen in the Mediterranean sea mussel ( Mytilus galloprovincialis ), a type of mussel widely distributed on the coast of Korea. The present invention was completed by grasping the molecular biological response of the mediterranean sea to a decrease in the amount of dissolved oxygen.

SUMMARY OF THE INVENTION An object of the present invention is to provide a microarray chip capable of stress detection or health diagnosis of marine ecosystems.

It is another object of the present invention to provide a stress detection or health diagnostic method for marine ecosystems.

Still another object of the present invention is to provide a kit for detecting or diagnosing stress in marine ecosystems.

In order to achieve the above object, the present invention provides a microarray chip for stress detection or health diagnosis of marine ecosystems, wherein the nucleic acid sequence oligonucleotide of any one or more genes selected from the group below or its complementary strand molecules are integrated;

60S ribosomal protein L32 protein mRNA, 40S ribosomal protein S3, 60S ribosomal protein L3 and related proteins, Ribosomal protein L11 mRNA, Elongation factor 2 mRNA, 40S ribosomal protein S2 putative mRNA / 30S ribosomal protein S5, 60S acidic ribosomal protein P2 (Genbank Accession FM165434.1), 40S ribosomal protein S4, Nucleolar complex associated 2 homolog, 40S ribosomal protein S28, 60S ribosomal protein L21, Elongation factor TU (tufA) gene, EF1-alpha mRNA for elongation factor 1 alpha (Genbank Accession No. EU684228.1), RecA gene, Zinc finger NFX1-type containing 1, tRNA-Leu (trnL) gene, Molecular chaperone (HSP90 family), Prefoldin subunit 1 putative mRNA, DnaJ domain containing protein mRNA, Hsp70-1 gene (Genbank Accession No. AJ585375.1), Aspartate beta-hydroxylase (ASPH), Matrix metallopeptidase 9, Heat shock protein 12B, C-type lectin mRNA, LIM protein (LIM) mRNA, Low-density lipoprotein receptors containing Ca2 + -binding EGF-like domains , Surfactant protein A, Calcium-dep endent protein kinase, Calcium / calmodulin-dependent protein kinase II alpha, GTPase-activating protein GYP 7, Ral-GDS related protein Rgr, CEGP1 protein, MEGF10 protein, 18S ribosomal RNA gene (Genbank Accession No. DQ640546.1), 16S ribosomal RNA gene (Genbank Accession No. AF023552.1), Fibrillar collagen, Phosphate regulatory protein genes phoB-phoR, Galactose-binding lectin, Lec4 lectin mRNA, Tubulin beta 2A, microtubule associated protein (RHAMM) mRNA , Chitinase mRNA, Sodium-dependent phosphate / anion cotransport, Endo-1,3-beta-glucanase Engl1, Organic anion transporter, Phosphoglycerate / bisphosphoglycerate mutase family protein, Peptidoglycan glycosyltransferase, ABC transporter AbcG16, Extracellular Cu / Zn-superoxide dismutase mRNA ( Genbank Accession No. FM177867.1), Immunoglobulin kappa chain complex (Igk), 6-deoxyerythronolyde B synthase II, Polyketide synthase gene cluster, Farnesyl diphosphate synthase, Gamma-glutamyltransferase 8 pseudogene, Cytochrome P-450 hydroxylase homolog (nidi) gene, Anthranilate N-methyltransferase mRNA, Mitochondrial ubiquinol-cytochrome c reductase iron-sulfur subunit, Cytochrome oxidase subunit I gene (Genbank Accession No. GQ480294.1), NADH: ubiquinone oxidored uctase NDUFA2 / B8 subunit, ATP synthase F0 subunit 6, Sn-glycerol-3-phosphate dehydrogenase, NADH dehydrogenase subunit 4 (Genbank Accession No. AF315186.1), Flavin containing monooxygenase 3 (FMO3) gene, Guanine nucleotide-binding protein subunit beta-like protein 1 (gnb1l) mRNA, Carcinolectin5b-5, Rhamnose-binding lectin, Ubiquitin-protein ligase E, Sialic acid binding lectin, Fertility restorer-like protein, Somatic embryogenesis receptor-like kinase 2, Transcriptional activator, TenA family, Transcription antiterminator, BglG family, Transcriptional regulator, LacI family, Leucine-rich repeat kinase 2, Transcriptional regulator, TetR family, SH2 domain containing 4B ( SH2D4B), Ecotype Hr-5 disease resistance protein, Collagen protein, MHC class II antigen beta (Genbank Accession No. AJ249992.1), Interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), Mytilin C precursor, Endoproteinase Arg-C precursor , Apextrin, Neuromacin, Matrix protein (gag), Opticin (Optc) gene, Glycopeptide AFGP polyprotein precursor gene, Cadherin 11 (Cdh11) gene, Microsatellite Myco-14 sequence, Proteasome beta 9 subunit gene, Hemagglutinin / ameboc yte aggregation factor precursor putative mRNA, Alpha 10 subunit of nicotinic acetylcholine receptor, MFN2 gene for mitofusin 2, Rendezvin (RDZ) mRNA, Calnexin (pp90) mRNA, Interleukin-20 receptor alpha, Interferon regulatory factor 7 (IRF7) mRNA, DAZ- associated protein 1 putative mRNA, Cytotactin and NADH -ubiquitin oxidoreductase chain 2.

In addition,

1) separating RNA from mediterranean mussels suspected of being exposed to the stress of the marine ecosystem of the experimental group and normal mediterranean mussels of the control group;

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 according to the present invention;

4) analyzing the reacted microarray chip; And

5) provides a method for detecting or diagnosing stress in a marine ecosystem including comparing the level of gene expression integrated in the microarray chip according to the present invention from the analyzed data with a control group.

In addition,

1) separating RNA from mediterranean mussels suspected of being exposed to the stress of the marine ecosystem of the experimental group and normal mediterranean mussels of the control group;

2) Quantitative real-time reverse transcript polymerase chain reaction (RT-PCR) using RNA pairs of step 1) using primer pairs complementary to genes integrated in the microarray chip according to the present invention and amplifying genes qRT-PCR); And

3) provides a method for detecting or diagnosing stress in a marine ecosystem comprising comparing the gene product of step 2) with a control to confirm the expression level.

In addition, the present invention provides a kit for stress detection or health diagnosis of the marine ecosystem comprising a microarray chip according to the present invention.

In addition, the present invention provides a kit for stress detection or health diagnosis of a marine ecosystem comprising a primer pair that is complementary to the gene integrated in the microarray chip according to the present invention and can amplify the gene.

Since the microarray chip according to the present invention accumulates the mediterranean seaweed gene corresponding to the decrease in dissolved oxygen amount, the microarray chip of the present invention, a diagnostic method using the same, and a kit including the microarray chip are used for detecting stress in marine ecosystems or It can be useful for medical examination. In addition, a diagnostic method using a primer pair that is complementary to a gene integrated in a microarray chip according to the present invention and amplifies the gene and a kit including the primer pair may be usefully used for stress detection or health diagnosis of marine ecosystems. have.

Figure 1 is a graph showing the difference in gene expression of mediterranean mussels cultured for 24 hours and 72 hours at 1 mg / L of dissolved oxygen (DO) dissolved oxygen. Gene fragments that appear specifically are indicated by arrows.
1: Mediterranean mussels incubated for 72 hours under normal conditions
2: experimental group, Mediterranean mussels exposed to free oxygen for 24 hours
3: experimental group, Mediterranean mussels exposed to empty oxygen for 72 hours
ACPX: Type of primer applied

Hereinafter, the present invention will be described in detail.

The present invention provides a microarray chip for stress detection or health diagnosis of marine ecosystems, in which nucleic acid sequence oligonucleotides or complementary strand molecules of any one or more genes selected from the following groups are integrated:

60S ribosomal protein L32 protein mRNA, 40S ribosomal protein S3, 60S ribosomal protein L3 and related proteins, Ribosomal protein L11 mRNA, Elongation factor 2 mRNA, 40S ribosomal protein S2 putative mRNA / 30S ribosomal protein S5, 60S acidic ribosomal protein P2, 40S ribosomal protein S4, Nucleolar complex associated 2 homolog, 40S ribosomal protein S28, 60S ribosomal protein L21, Elongation factor TU (tufA) gene, EF1-alpha mRNA for elongation factor 1 alpha, RecA gene, Zinc finger NFX1-type containing 1, tRNA- Leu (trnL) gene, Molecular chaperone (HSP90 family), Prefoldin subunit 1 putative mRNA, DnaJ domain containing protein mRNA, Hsp70-1 gene, Aspartate beta-hydroxylase (ASPH), Matrix metallopeptidase 9, Heat shock protein 12B, C-type lectin mRNA, LIM protein (LIM) mRNA, Low-density lipoprotein receptors containing Ca2 + -binding EGF-like domains, Surfactant protein A, Calcium-dependent protein kinase, Calcium / calmodulin-dependent protein kinase II alpha, GTPase-activating protein G YP 7, Ral-GDS related protein Rgr, CEGP1 protein, MEGF10 protein, 18S ribosomal RNA gene, 16S ribosomal RNA gene, Fibrillar collagen, Phosphate regulatory protein genes phoB-phoR, Galactose-binding lectin, Lec4 lectin mRNA, Tubulin beta 2A, microtubule associated protein (RHAMM) mRNA, Chitinase mRNA, Sodium-dependent phosphate / anion cotransport, Endo-1,3-beta-glucanase Engl1, Organic anion transporter, Phosphoglycerate / bisphosphoglycerate mutase family protein, Peptidoglycan glycosyltransferase, ABC transporter AbcG16, Extracellular Cu / Zn-superoxide dismutase mRNA, Immunoglobulin kappa chain complex (Igk), 6-deoxyerythronolyde B synthase II, Polyketide synthase gene cluster, Farnesyl diphosphate synthase, Gamma-glutamyltransferase 8 pseudogene, Cytochrome P-450 hydroxylase homolog (nidi) gene, Anthranilate N -methyltransferase mRNA, Mitochondrial ubiquinol-cytochrome c reductase iron-sulfur subunit, Cytochrome oxidase subunit I gene, NADH: ubiquinone oxidoreductase NDUFA2 / B8 subunit, ATP synthase F0 subunit 6, Sn-glycerol-3-phosphate dehydrogenase, NADH dehydrogenase subunit 4, Flavin containing monooxygenase 3 (FMO3) gene, Guanine nucleotide-binding protein subunit beta-like protein 1 (gnb1l) mRNA, Carcinolectin5b-5, Rhamnose -binding lectin, Ubiquitin-protein ligase E, Sialic acid binding lectin, Fertility restorer-like protein, Somatic embryogenesis receptor-like kinase 2, Transcriptional activator, TenA family, Transcription antiterminator, BglG family, Transcriptional regulator, LacI family, Leucine-rich repeat kinase 2, Transcriptional regulator, TetR family, SH2 domain containing 4B (SH2D4B), Ecotype Hr-5 disease resistance protein, Collagen protein, MHC class II antigen beta, Interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), Mytilin C precursor , Endoproteinase Arg-C precursor, Apextrin, Neuromacin, Matrix protein (gag), Opticin (Optc) gene, Glycopeptide AFGP polyprotein precursor gene, Cadherin 11 (Cdh11) gene, Microsatellite Myco-14 sequence, Prot easome beta 9 subunit gene, Hemagglutinin / amebocyte aggregation factor precursor putative mRNA, Alpha 10 subunit of nicotinic acetylcholine receptor, MFN2 gene for mitofusin 2, Rendezvin (RDZ) mRNA, Calnexin (pp90) mRNA, Interleukin-20 receptor alpha, Interferon regulatory factor 7 (IRF7) mRNA, DAZ-associated protein 1 putative mRNA, Cytotactin and NADH -ubiquitin oxidoreductase chain 2.

It is preferable that the microarray chip is a microarray chip for stress detection or health diagnosis of marine ecosystems in which nucleic acid sequence oligonucleotides or complementary strand molecules of any one or more genes selected from the following groups are integrated, but is not limited thereto.

18S ribosomal RNA (Genbank Accession No. DQ640546.1), ALPHA 10 nAChR gene for alpha 10 subunit of nicotinic acetylcholine, EF1-alpha mRNA for elongation factor 1 alpha (Genbank Accession No. EU684228.1), chitinase mRNA, cytochrome oxidase subunit 1 (cox1) (Genbank Accession No. GQ480294.1), NADH dehydrogenase 1 alpha subcomplex subunit 2 putative mRNA, calcium / calmodulin-dependent protein kinase II alpha, GTPase-activating protein GYP 7, phosphoglycerate / bisphosphoglycerate mutase family protein, hsp90, ATP synthase F0 subunit 6.

In the microarray chip, the gene is mediterranean mussel ( Mytilus galloprovincialis ), but is not limited thereto.

In the microarray chip, the gene is preferably increased or decreased by changing the amount of dissolved oxygen, but is not limited thereto.

In the microarray chip, the stress is preferably a change in the amount of dissolved oxygen in seawater, but is not limited thereto. The amount of dissolved oxygen in the seawater is preferably 3 mg / L or less, and more preferably 1 mg / L or less, but is not limited thereto. The change in the amount of dissolved oxygen in the seawater is preferably due to the formation of poor oxygen (hypoxia, low oxygen) water mass, but is not limited thereto.

In the microarray chip, it is preferable to detect a marine ecosystem response according to a change in dissolved oxygen amount, but is not limited thereto.

In a specific embodiment of the present invention, it was confirmed that 100 genes whose expression is increased or decreased by decreasing the amount of dissolved oxygen compared to the control group, and the nucleic acid sequence described in SEQ ID NOS: 1 to 100 through sequencing analysis (Fig. 1). .

Also,

1) separating RNA from mediterranean mussels suspected of being exposed to the stress of the marine ecosystem of the experimental group and normal mediterranean mussels of the control group;

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 according to the present invention;

4) analyzing the reacted microarray chip; And

5) provides a method for detecting or diagnosing stress in a marine ecosystem including comparing the level of gene expression integrated in the microarray chip according to the present invention from the analyzed data with a control group.

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). It is not limited to this.

In the method, the stress is preferably a change in the amount of dissolved oxygen in seawater, but is not limited thereto. The amount of dissolved oxygen in the seawater is preferably 3 mg / L or less, and more preferably 1 mg / L or less, but is not limited thereto. The change in the amount of dissolved oxygen in the seawater is preferably due to the formation of poor oxygen (hypoxia, low oxygen) water mass, but is not limited thereto.

In the above method, it is preferable to detect a marine ecosystem response due to a change in dissolved oxygen amount, but is not limited thereto.

Also,

1) separating RNA from mediterranean mussels suspected of being exposed to the stress of the marine ecosystem of the experimental group and normal mediterranean mussels of the control group;

2) Quantitative real-time reverse transcript polymerase chain reaction (RT-PCR) using RNA pairs of step 1) using primer pairs complementary to genes integrated in the microarray chip according to the present invention and amplifying genes qRT-PCR); And

3) provides a method for detecting or diagnosing stress in a marine ecosystem comprising comparing the gene product of step 2) with a control to confirm the expression level.

In the method, the stress is preferably a change in the amount of dissolved oxygen in seawater, but is not limited thereto. The amount of dissolved oxygen in the seawater is preferably 3 mg / L or less, and more preferably 1 mg / L or less, but is not limited thereto. The change in the amount of dissolved oxygen in the seawater is preferably due to the formation of poor oxygen (hypoxia, low oxygen) water mass, but is not limited thereto.

In the above method, it is preferable to detect a marine ecosystem response due to a change in dissolved oxygen amount, but is not limited thereto.

In another aspect, the present invention provides a kit for stress detection or health diagnosis of a marine ecosystem comprising a microarray chip according to the present invention.

In the kit, the stress is preferably a change in the amount of dissolved oxygen in seawater, but is not limited thereto. The amount of dissolved oxygen in the seawater is preferably 3 mg / L or less, and more preferably 1 mg / L or less, but is not limited thereto. The change in the amount of dissolved oxygen in the seawater is preferably due to the formation of poor oxygen (hypoxia, low oxygen) water mass, but is not limited thereto.

In the kit, it is preferable to detect a marine ecosystem response due to a change in dissolved oxygen amount, but is not limited thereto.

The kit further comprises any one selected from the group consisting of a streptavidin-alkali dephosphorase conjugate (strepavidin-like phosphatease conjugate), a chemical fluorescence (chemiflurorensce) and a chemiluminescent (chemiluminescent) group Preferred but not limited thereto.

In the kit, any one selected from the group of buffers used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, dNTPs and rNTPs (premixed or separated feed), labeling reagents, and wash buffers. It is preferable to further include, but is not limited thereto.

In another aspect, the present invention provides a kit for stress detection or health diagnostics of marine ecosystems comprising primer pairs complementary to the genes integrated in the microarray chip according to the present invention and capable of amplifying the genes.

In the kit, the stress is preferably a change in the amount of dissolved oxygen in seawater, but is not limited thereto. The amount of dissolved oxygen in the seawater is preferably 3 mg / L or less, and more preferably 1 mg / L or less, but is not limited thereto. The change in the amount of dissolved oxygen in the seawater is preferably due to the formation of poor oxygen (hypoxia, low oxygen) water mass, but is not limited thereto.

In the kit, it is preferable to detect a marine ecosystem response due to a change in dissolved oxygen amount, but is not limited thereto.

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.

Mediterranean sea  Culture and Empty oxygen  Condition exposure

<1-1> Mediterranean sea  culture

Mediterranean mussels harvested from Jangmok Bay, Geoje-si, Gyeongnam, Korea, were put in a net of 10 to 15 individuals, and cultivated on hooks installed in a tank. The seawater used natural seawater which passed through three kinds of filters (100, 10, and 1 micrometer). The water temperature was fixed at 20-21 ° C. using an underwater heater and incubated for at least one week. Sufficient air was supplied using an air supply during the incubation period. Cultivation was carried out in dark conditions without light.

<1-2> Mediterranean sea Empty oxygen  Condition exposure

The Mediterranean mussels of Example <1-1> were incubated for 3 days with a fixed amount of dissolved oxygen at 1 mg / L using nitrogen gas and an air supply. Samples were taken at 24 and 72 hours. The water temperature was maintained at 20-21 ° C. Dissolved oxygen content was adjusted using AquaController (Neptune Systems, San Jose, Calif., USA).

Empty oxygen  Genetic change measurement by conditional exposure

<2-1> RNA Separation of

TRI Reagent solution (Molecular Research Center Inc, Cincinnati, Ohaio, USA) 1 to 50-100 mg of mediterranean sea gill tissue of the test group and the unexposed control group exposed to the hypoxic conditions obtained in Example <1-2> ㎖ was added, homogenized using a glass homogenizer, and allowed to stand at room temperature for 5 minutes. 200 µl of chloroform was added and mixed well, and the mixture was left at room temperature for 10 minutes and centrifuged (12,000 x g, 4 ° C) for 15 minutes. The supernatant was taken, 500 μl of isopropanol was added, and the mixture was allowed to stand at room temperature for 5 minutes. After centrifugation (12,000 x g, 4 degreeC) for about 20 minutes, the solution was removed and only the precipitate was taken. 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 DEPC-treated water.

<2-2> cDNA  synthesis

GeneFishing ^TM DEG kits (Seegene, Korea) were used to isolate specific genes corresponding to changes in dissolved oxygen. The GeneFishing ^™ DEG kit was developed for the isolation of PCR-based differential expression genes and is based on the Differential display (DD) -PCR method designed by Liang and Pardee (Science 1992, 257: 967-971). The DD-PCR technique has the advantage of being able to isolate differentially expressed genes from various experimental groups with a small amount of RNA, but has the disadvantage of detecting false-positive results with a relatively high frequency. Various attempts have been made to remedy these shortcomings, and recently, the GeneFishing ^TM DEG kit has been used to increase specificity using a specially designed annealing control primer system. The cDNA of the control group and the experimental group was synthesized using mRNA (3.0 μg) purified from the extracted total RNA as a template, dT-ACP1 as a primer, and MMLV RT-ase as a reaction enzyme.

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

PCR was carried out using the first strand cDNA synthesized as a primer and dT-ACP2 and 120 kinds of annealing control primers (ACPs) as primers. The PCR conditions were 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 performed at 72 degreeC for 5 minutes. 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 plasmid was extracted. Sequence analysis was performed.

As a result, as shown in FIG. 1, 100 genes whose expression was increased or decreased by decreasing the amount of dissolved oxygen compared to the control group were identified, and the nucleic acid sequences described in SEQ ID NOs: 1 to 100 were identified through sequence analysis.

60S ribosomal protein L32 protein mRNA (SEQ ID NO: 1), 40S ribosomal protein S3 (SEQ ID NO: 2), 60S ribosomal protein L3 and related proteins (SEQ ID NO: 3), Ribosomal protein L11 mRNA (SEQ ID NO: 4), Elongation factor 2 mRNA (SEQ ID NO: 5), 40S ribosomal protein S2 putative mRNA / 30S ribosomal protein S5 (SEQ ID NO: 6), 60S acidic ribosomal protein P2 (Genbank Accession No. FM165434.1) (SEQ ID NO: 7), 40S ribosomal protein S4 (SEQ ID NO: 8), Nucleolar complex associated 2 homolog (SEQ ID NO: 9), 40S ribosomal protein S28 (SEQ ID NO: 10), 60S ribosomal protein L21 (SEQ ID NO: 11), Elongation factor TU (tufA) gene (SEQ ID NO: 12), EF1-alpha mRNA for elongation factor 1 alpha (Genbank Accession No. EU684228.1) (SEQ ID NO: 13), RecA gene (SEQ ID NO: 14), Zinc finger NFX1-type containing 1 ( SEQ ID NO: 15), tRNA-Leu (trnL) gene (SEQ ID NO: 16), Molecular chaperone (HSP90 family) (SEQ ID NO: 17), Prefoldin subunit 1 putative mRNA (SEQ ID NO: 18), DnaJ domai n containing protein mRNA (SEQ ID NO: 19), Hsp70-1 gene (Genbank Accession No. AJ585375.1) (SEQ ID NO: 20), Aspartate beta-hydroxylase (ASPH) (SEQ ID NO: 21), Matrix metallopeptidase 9 (SEQ ID NO: No. 22), Heat shock protein 12B (SEQ ID NO: 23), C-type lectin mRNA (SEQ ID NO: 24), LIM protein (LIM) mRNA (SEQ ID NO: 25), Low-density lipoprotein receptors containing Ca2 + -binding EGF-like domains (SEQ ID NO: 26), Surfactant protein A (SEQ ID NO: 27), Calcium-dependent protein kinase (SEQ ID NO: 28), Calcium / calmodulin-dependent protein kinase II alpha (SEQ ID NO: 29), GTPase activating protein GYP 7 (SEQ ID NO: 30), Ral-GDS related protein Rgr (SEQ ID NO: 31), CEGP1 protein (SEQ ID NO: 32), MEGF10 protein (SEQ ID NO: 33), 18S ribosomal RNA gene (Genbank Accession No. DQ640546.1) (SEQ ID NO: 34), 16S ribosomal RNA gene (Genbank Accession No. AF023552.1) (SEQ ID NO: 35), Fibrillar collagen (SEQ ID NO: 36), Phosphate regulatory protein genes phoB-phoR (SEQ ID NO: : 37), Galactose-binding lectin (SEQ ID NO: 38), Lec4 lectin mRNA (SEQ ID NO: 39), Tubulin beta 2A (SEQ ID NO: 40), microtubule associated protein (RHAMM) mRNA (SEQ ID NO: 41), Chitinase mRNA (SEQ ID NO: 42), Sodium-dependent phosphate / anion cotransport (SEQ ID NO: 43), Endo-1,3-beta-glucanase Engl1 (SEQ ID NO: 44), Organic anion transporter (SEQ ID NO: 45), Phosphoglycerate / bisphosphoglycerate mutase family protein (SEQ ID NO: 46), Peptidoglycan glycosyltransferase (SEQ ID NO: 47), ABC transporter AbcG16 (SEQ ID NO: 48), Extracellular Cu / Zn-superoxide dismutase mRNA (Genbank Accession No. FM177867.1) (SEQ ID NO: No. 49), Immunoglobulin kappa chain complex (Igk) (SEQ ID NO: 50), 6-deoxyerythronolyde B synthase II (SEQ ID NO: 51), Polyketide synthase gene cluster ( SEQ ID NO: 52), Farnesyl diphosphate synthase (SEQ ID NO: 53), Gamma-glutamyltransferase 8 pseudogene (SEQ ID NO: 54), Cytochrome P-450 hydroxylase homolog (nidi) gene (SEQ ID NO: 55), Anthranilate N-methyltransferase mRNA (SEQ ID NO: 56), Mitochondrial ubiquinol-cytochrome c reductase iron-sulfur subunit (SEQ ID NO: 57), Cytochrome oxidase subunit I gene (Genbank Accession No. GQ480294.1) (SEQ ID NO: 58), NADH: ubiquinone oxidoreductase NDUFA2 / B8 subunit (SEQ ID NO: 59), ATP synthase F0 subunit 6 (SEQ ID NO: 60), Sn-glycerol-3-phosphate dehydrogenase (SEQ ID NO: 61), NADH dehydrogenase subunit 4 (Genbank Accession No. AF315186.1) (SEQ ID NO: 62), Flavin containing monooxygenase 3 (FMO3) gene (SEQ ID NO: 63), Guanine nucleotide-binding protein subunit beta-like protein 1 ( gnb1l) mRNA (SEQ ID NO: 64), Carcinolectin5b-5 (SEQ ID NO: 65), Rhamnose-binding lectin (SEQ ID NO: 66), Ubiquitin-protein ligase E (SEQ ID NO: 67), Sialic acid binding lectin (SEQ ID NO: : 68), Fertility restorer-like protein (SEQ ID NO: 69), Somatic embryogenesis receptor-like kinase 2 (SEQ ID NO: 70), Transcriptional activator TenA family (SEQ ID NO: 71), Transcription antiterminator BglG family (SEQ ID NO: 72) ), Transcriptional regulator LacI family (SEQ ID NO: 73), Leucine-rich repeat kinase 2 (SEQ ID NO: 74), Transcriptional regulator TetR family No. 75), SH2 domain containing 4B (SH2D4B) (SEQ ID NO: 76), Ecotype Hr-5 disease resistance protein (SEQ ID NO: 77), Collagen protein (SEQ ID NO: 78), MHC class II antigen beta (Genbank Accession No. AJ249992.1) (SEQ ID NO: 79), Interleukin 1 receptor accessory protein-like 1 (IL1RAPL1) (SEQ ID NO: 80), Mytilin C precursor (SEQ ID NO: 81), Endoproteinase Arg-C precursor (SEQ ID NO: 82) , Apextrin (SEQ ID NO: 83), Neuromacin (SEQ ID NO: 84), Matrix protein (gag) (SEQ ID NO: 85), Opticin (Optc) gene (SEQ ID NO: 86), Glycopeptide AFGP polyprotein precursor gene (SEQ ID NO: 87), Cadherin 11 (Cdh11) gene (SEQ ID NO: 88), Microsatellite Myco-14 sequence (SEQ ID NO: 89), Proteasome beta 9 subunit gene (SEQ ID NO: 90), Hemagglutinin / amebocyte aggregation factor precursor putative mRNA (SEQ ID NO: 87) 91: Alpha 10 subunit of nicotinic acetylcholine receptor (SEQ ID NO: 92), MFN2 gene for mitofusin 2 (SEQ ID NO: 93), Rendezvin (RDZ) mRNA (SEQ ID NO: 94), Calnexin (pp90) mRNA (SEQ ID NO: 91) 95: Interleukin-20 receptor alpha (SEQ ID NO: 96), Interferon regulatory factor 7 (IRF7) mRNA (SEQ ID NO: 97), DAZ-associated protein 1 putative mRNA (SEQ ID NO: 98), Cyto tactin (SEQ ID NO: 99) and NADH -ubiquitin oxidoreductase chain 2 (SEQ ID NO: 100).

Quantitative Analysis of Changes in Expression Levels of Selected Genes

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

Of the 100 genes isolated in Example <2-3>, the following 11 genes, which are deeply related to the self-defense mechanism against external stress, were selected.

18S ribosomal RNA gene, ALPHA 10 nAChR gene for alpha 10 subunit of nicotinic acetylcholine, EF1-alpha mRNA for elongation factor 1 alpha, chitinase mRNA, cytochrome oxidase subunit 1 (cox1) gene, NADH dehydrogenase 1 alpha subcomplex subunit 2 putative mRNA, calcium / calmodulin-dependent protein kinase II alpha, GTPase-activating protein GYP 7, phosphoglycerate / bisphosphoglycerate mutase family protein, hsp90, ATP synthase F0 subunit 6.

For these, primers for real-time quantitative PCR (qPCR) were designed and synthesized (Table 1).

Primer sequencing of target genes for real-time quantitative PCR gene Base sequence of primer 18S ribosomal RNA gene F: 5'-GGTCGGTGAAACATCAATC-3 '(SEQ ID NO: 101)
R: 5'-AGAATACAAGTACCGAAAGG-3 '(SEQ ID NO: 102) ALPHA 10 nAChR gene F: 5'-GGTACTAGCGTTGGTATCATC-3 '(SEQ ID NO: 103)
R: 5'-GTCCGCTTTTACTGTTTTTC-3 '(SEQ ID NO: 104) EF1-alpha mRNA F: 5'-GTGGGATGATGTTGATATAG-3 '(SEQ ID NO: 105)
R: 5'-CCATTGCCTATAGCATTTAC-3 '(SEQ ID NO: 106) Chitinase mRNA F: 5'-CAGGGAAGTTGTAATATCTG-3 '(SEQ ID NO: 107)
R: 5'-TACTCTGAAAACACAGGTGAC-3 '(SEQ ID NO: 108) Cytochrome oxidase subunit 1 (cox1) gene F: 5'-GTGTCTTCTTATGGGTCTG-3 '(SEQ ID NO: 109)
R: 5'-GCTATAAACATGCTTTCTCC-3 '(SEQ ID NO: 110) NADH dehydrogenase 1 alpha subcomplex subunit 2 putative mRNA F: 5'-TGGACAGAGTTTAAAGGAG-3 '(SEQ ID NO: 111)
R: 5'-CTAGCATAGACTTTTGGTTG-3 '(SEQ ID NO: 112) Calcium / calmodulin-dependent protein kinase II alpha F: 5'-ACAGTCGGCACAGGTACAC-3 '(SEQ ID NO: 113)
R: 5'-GCATACTACGCTCTACACTTC-3 '(SEQ ID NO: 114) GTPase-activating protein GYP 7 F: 5'-CAGGTTTTACTGCTACAGG-3 '(SEQ ID NO: 115)
R: 5'-GTTAATCACGATCCCTTGTG-3 '(SEQ ID NO: 116) Phosphoglycerate / bisphosphoglycerate mutase family protein F: 5'-GGTGACTGTTTTTATGGTC-3 '(SEQ ID NO: 117)
R: 5'-CGAAGAGAATGAAATGTCC-3 '(SEQ ID NO: 118) Hsp90 F: 5'-GGTTGCTGATAAAGTAGTTG-3 '(SEQ ID NO: 119)
R: 5'-ATTCAGTCTGGTCTTCTTTC-3 '(SEQ ID NO: 120) ATP synthase F0 subunit 6 F: 5'-ATATGTAGAAGGGAGTTGG-3 '(SEQ ID NO: 121)
R: 5'-ATCTAACTCCTATGGTCCTC-3 '(SEQ ID NO: 122)

<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 μg of RNA was aliquoted and titrated to 9 by adding distilled water. Add 10 μl of 2 × RT buffer and 1 μl of 20 × Enzyme Mix, mix well, spin down, and react 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 μl (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. PCR was carried out under the conditions of 95 ° C for 10 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds.

The CT values of the genes of interest (a-tubulin) and genes of interest for each sample were compared, and relative expression curves were quantified as relative values by a relative standard curve method (Table 2).

Real-time quantitative PCR  By analysis Empty oxygen  Expression level change of target gene by conditional exposure gene Exposure time  24 hours 72 hours 18S ribosomal RNA gene 22.02 reduction 5.94 increase ALPHA 10 nAChR gene 13.68 reduction 1.50 increase EF1-alpha mRNA 10.66 reduction 4.44 reduction Chitinase mRNA 10.71 reduction 2.91 reduction Cytochrome oxidase subunit 1 (cox1) gene 8.20 reduction 1.18 increase NADH dehydrogenase 1 alpha subcomplex subunit 2 putative mRNA 29.23 reduction Not detected Calcium / calmodulin-dependent protein kinase II alpha 52.6 reduction 1.55 reduction GTPase-activating protein GYP 7 2.01 reduction 3.05 reduction Phosphoglycerate / bisphosphoglycerate mutase family protein 344.83 reduction 3.09 reduction Hsp90 18.18 reduction 1.89 reduction ATP synthase F0 subunit 6 107.45 increase 6.82 reduction

The Mediterranean sea mussel gene corresponding to the decrease in dissolved oxygen amount of the present invention can be usefully used as a biosensor for monitoring and diagnosing the decrease in dissolved oxygen amount and the marine ecosystem status according to the empty oxygen condition specific gene. It can be used as biomarkers and sensors that can predict the pathological phenomena that may occur in the future by specifying physiological changes, and can be effectively used for stress detection or health diagnosis of marine ecosystems.

<110> Korea Ocean Research and Development Institute <120> Dissolved oxygen change responsive genes in Mytilus          galloprovincialis and the method for diagnosing marine ecosystem          using the same <130> 11p-04-12 <160> 122 <170> Kopatentin 2.0 <210> 1 <211> 264 <212> DNA <213> Mytilus galloprovincialis <400> 1 aaatattggt tatggatcaa ataagaaaac caaacacttg atgcctgatg gtttccaaaa 60 attccttgta cataatgtta aggaattgga agtccttttg atgcagaata gaacatttgc 120 agctgagatt gcacacaatg tttccagcag gaaaaggaaa gaaatcgttg agcgtgctca 180 gcagttgtct atcaaagtga ccaatccaaa tgctagactg agatcagagg acgatgaata 240 gaaaaataaa tgtggctcaa taaa 264 <210> 2 <211> 642 <212> DNA <213> Mytilus galloprovincialis <400> 2 tgtaacgaac cgcgcgttgt gtgcaactgc gcaagcagaa tcggttcgtt ataaattgat 60 ggagggtctt ccagtacgca gggcttgtta cggagttttg cgttatgtta tggcaaacgg 120 agcgaaggga gtcgaagtta tcgtctccgg gaaattaaga gcgcaacgcg ccaaatccat 180 gaaattcagc gatggttata tgatcaattc aggaaacgca aaggaattgt atatcacgga 240 agcggtccgc gatgttatgc tcaaacaagg agttatcggc attaaagtcc gtatccaatt 300 accttacgat ccttctggtg tcgacggatg ctcggttcct ctttctgatg tagtggaagt 360 tcatgaaccg aaagttttag accccgtaat tcatttttcc aagtttcgat ccgagcagtc 420 agaagaacaa ccttctgtag tcattcctga agaagagcat caatcttctt atccagaatc 480 cggtatctta cacccttcgg atcaaaatca gcaacaacag cagtatttcg ctgctcagca 540 acaagatgct gatttttcgc agtcttataa tagataaaag tgtgttggtt gtttggaatc 600 ttttctattg attctatttt ggattatatt gtgtgtttgg tt 642 <210> 3 <211> 284 <212> DNA <213> Mytilus galloprovincialis <400> 3 tatggagaag tatccaacga ctttgtcatg gtaaagggat gttgtgttgg acccaagaag 60 agagttgtca cacttagaaa gtcttttttg gcatcattca aaaagaagca aatggagaag 120 attgacctga aattcattga cactgccagt aaatttggtc atggacgttt ccagacattc 180 aaggagaaac acaactttat gggtccattg aaaaaagatg ctgtagcaaa aacagaggct 240 ttttagaaat gggacatttc aaatgaataa aaaatctgtg atgt 284 <210> 4 <211> 463 <212> DNA <213> Mytilus galloprovincialis <400> 4 gtgcttgaac agctaactgg tcagacccca gtgctctcaa aagcacgtta taccgtcagg 60 tccttcggta tcagaagaaa tgaaactatt gcctgtcact gtacagtacg aggcgctaag 120 gcagaagaga tcttagaaaa aggtttgaag gttagagaat acgagttgag gaaaaataac 180 ttttcccaaa ctggcaactt tggttttggt attcaggaac acattgattt aggaattaag 240 tatgatcctg gaattggtat ctatggtatg gatttctaca tcgtgctagg aagagcaggc 300 ttcaacatcg cacagagacg tagaaggaag agcaagattg gaccaaacca taaagtttat 360 aaagaagaat caatgaaatg gttccaacaa aagtatgatg gaattattct gcctggaaaa 420 taaaggacta gaaattaaaa aaataaaacc tggctgtgat cag 463 <210> 5 <211> 111 <212> DNA <213> Mytilus galloprovincialis <400> 5 ttacatggtt gggctttcac tttgaaagac tttgctgcta tgtacacaaa gaaatttggc 60 attgctgagg acaaactgat gaagagattg tggggtgaca atttctacaa c 111 <210> 6 <211> 220 <212> DNA <213> Mytilus galloprovincialis <400> 6 caaggccaca tatgcagcta tagccaatac ctatttttac ctgactcctg atttgtggaa 60 ggaaaccacc ttcaccaagt ctccatatca ggaattcact gattatctgg ccaagaatca 120 tacaagagaa gcaggccaga aacaagaagt caaagtttac taggactttt tgaacacaat 180 agatacattg tatcagaaat aaactgaata catatatata 220 <210> 7 <211> 352 <212> DNA <213> Mytilus galloprovincialis <400> 7 gtctaccagg cattcgcttc atgctttcgg ccgtttgact tgatcaaaga agttgttcac 60 agtttcgtgt ctgaaagact taaaaatgcg ttacgtggca gcttacctac ttgcatccct 120 aggaggaaac acaaatccat cagccagtga ccttgaaaag attattgggt cagttggaat 180 tgaagccgaa gctgataaga taaagaaaat tataagtgaa ctcaaaggaa aaaaccttga 240 agacctgatc aaagaaggtc aagaaaagct ggcctcagta cctgcaggag gagctgttgc 300 tgctgcacct gctgctgccg ttgctgctga agcacctaaa ggaggagcaa ag 352 <210> 8 <211> 460 <212> DNA <213> Mytilus galloprovincialis <400> 8 aagtctgaag aagcaaagta caaactgtgc cgtgtaaaga aagttggagt agctttgaag 60 ggagtttgct atgttgtcac acatgacgga cgtacaattc gttatccaga tccattggtc 120 aaagtcaacg acacaataat ggttgacatt gcaactggaa agattaaaga tttcatcaag 180 tttgattcag gaaatctctg tatgataact ggaggccata atttgggaag agttggagtt 240 attcagcaca gagaaagaca tccaggatca ttcgacattg ttcacattaa agattcttta 300 ggacatagtt ttgctaccag attaagttac gttttcatca tcggaaaagg aaacaaacca 360 tatgtgtcac ttccacgtgg taaaggtgtc aaattgagta ttgctgaaga gagagatcgc 420 agaatggcag ctaaataaac tgttagagac tgcatcaaat 460 <210> 9 <211> 130 <212> DNA <213> Mytilus galloprovincialis <400> 9 caagaaaagg aaaatggaag agttgagtgt tgatgagttt atgttaggag actttgacag 60 tgacagtgac aaagaagata cagctgttca ccaaaaacag aatcagaaga aaagaaaaaa 120 tgacggactt 130 <210> 10 <211> 279 <212> DNA <213> Mytilus galloprovincialis <400> 10 tggataaact ccagccagta aaacttgcta aagtcaccaa aattttgggc agaacaggat 60 cacaaggaca gtgtactcag gtccgtgtag aatttttaga tgatagtaac agatccatca 120 tcagaaatgt taaaggtcca gtaagagaag gagatatcct gacactgtta gaatctgaga 180 gagtagccag aagattaagg taaccacaga caaacaagaa atggtgaagt ggttctacag 240 ataggactgt atagtctgaa taaatcttag aaaaccctc 279 <210> 11 <211> 100 <212> DNA <213> Mytilus galloprovincialis <400> 11 agccagattt gtgaggacaa aatataacaa gccacagatc ttagaaccta taccctatga 60 atttattgca taaggactta taataaagtg gtctgagatg 100 <210> 12 <211> 495 <212> DNA <213> Mytilus galloprovincialis <400> 12 gtctaccagg cattcgcttc atggggggca tccagcatga ctgattgtct tgcaatttgt 60 gcagaatctc gaaaatgcgt agcaattaca ttttgcaaga cacaggccga gtgtccaagt 120 tgggcacaca atgtgtgtta catgtataat aattatctta aggacggaat aacggctgag 180 cagtcaagta gctgggaaag tattgtattt acgtacaaga tagatacaac cgacagattt 240 gcgttaaatg atacaaccat tgtaggaact ccacaatccg ataacggtac aatagtttca 300 gaaacggcat gcaaaacaaa atgtgaaaat gatgcttatt gcgaggtgta ttcattctgt 360 tcatgtccgg ataaaagcat aaaatgtaaa ttgtatgccg gagaaagggt tattgcatta 420 actgaagaaa ttggaactaa aaccgaattt attcctccga aatgaagagt atattattaa 480 atactattaa atcaa 495 <210> 13 <211> 236 <212> DNA <213> Mytilus galloprovincialis <400> 13 agcaaaggcc agtggcaaga aatgatgtga ggcgtgggat gatgttgata tagaaatgaa 60 gaccgaaata aaaaacaaac taattttaag tcaaaattta agaccacagc aaattatgta 120 ttggacaatt tggttgctta gaagtaaaat tcatcgtaaa gaaatcaggc ggaaaaactt 180 gtaaatgcta taggcaatgg cataatttta tttatgttaa aaataaaaaa ataatt 236 <210> 14 <211> 494 <212> DNA <213> Mytilus galloprovincialis <400> 14 aaggagtaac cgatgtggta cttggttaag gcataggcca tgcactgctg ctaagtgtag 60 tgatccttca agaggaaaac cattttattt tgggacaact agtttgttgt gtatggcgaa 120 aggaaaacgc gaatcgtcac catctcgacc tacatgggca ctgacacaca gatggatcga 180 atacaatggg catttttttg aacgattaaa gacgggagga gatgtttata gctcgaattt 240 tcctcaggac tcgcaaaaat gttcttccaa gcgtgaaggc tttcctgccg gatacagcag 300 tctgggtata gattgtttga aacgttgtac aaataagtac agagatcatt atggtaaata 360 tcgtgtgcta acaaataact gccacgactt tgctaatcgg ttctctgata tgttatgcag 420 cagaaccacg tgtccttcgt ggtgttctta aattgaaacg attatctcct taaatattat 480 taaatgtgac ttct 494 <210> 15 <211> 225 <212> DNA <213> Mytilus galloprovincialis <400> 15 aatcaatcga agaagacaca ctgccatttc agcaatatat tgttcattgc aatagttcag 60 ttgatatgcc agattatctt gcttttgacc cggatgtaag atttgattta agatcgatac 120 tatctataaa tcaggcaaaa aatgagttaa gtgaacgttc ggtagaagat gtagacgatt 180 tactagtggc aatgaggaag catgtgcaaa gatcagcatt tatag 225 <210> 16 <211> 180 <212> DNA <213> Mytilus galloprovincialis <400> 16 aaaacagaag aagaaaaaag ataaaaagat gatagataaa ataaagacga actaagaaat 60 agtaatatgg aaaagtgctt gatttcaatt ttgagttatc aattaaattc aattgttggt 120 atttatcgaa ttgatgtgtt gtgcgtgtat gtatataaat aaaaacaatg tataaacttc 180                                                                          180 <210> 17 <211> 227 <212> DNA <213> Mytilus galloprovincialis <400> 17 tggagctgat atttctatga ttggacagtt tggtgtaggt ttctactccg cctacctggt 60 tgctgataaa gtagttgtcc agacaaggaa caacgatgat gaggaatata tctgggaatc 120 ggcagctggg ggatcattta cagtcaaaac ggtatcaggt ggtgagtctg tcggcagagg 180 taccaagatt acactttaca tgaaagaaga ccagactgaa tatttgg 227 <210> 18 <211> 293 <212> DNA <213> Mytilus galloprovincialis <400> 18 agatagaggc tcttaagaga acaattcaac attcgaaatt ggtatatcat gaactgtcat 60 ccctggcaga agaaacccgt ctgtatgaag gagttggtag aatgttcata ttgcagcctc 120 caaaagaaat taaaaaccag ttagattcaa agatgaaagc atcagaagaa aaaatagaat 180 ccttacagaa aaacaagaca tatttagaaa gaaatataaa agaaagtgag gacaatctta 240 gagaaatgat aacaagtcgt caaatggcaa aataaataaa ctgattacat cat 293 <210> 19 <211> 601 <212> DNA <213> Mytilus galloprovincialis <400> 19 aggtgactga cgaaattttt ctaggacttt cttatccttt atatttattt gacgcatttc 60 gaaaatgacc tccaatctga ttctagctgg acttggtata gcagcagtgg gttttggagg 120 gagatatcta accaggcatg caaaaactgt aacacagaca ctgaagaaac agatggatgc 180 atctggggcc ttgagcaagt actataaagg aggatttgac gcaaaaatgt caaaaagaga 240 ggctggttta attttaggag taagtccatc agcaagtaaa gtcaaggtga aagatgcaca 300 caaacgaata atgttattaa atcatccaga cagaggtggt tcaccatatc tggcatcaaa 360 gattaatgaa gcaaaggact attttgacag taacaaggca tagagataat gtgatctttc 420 cagtaccaca gtctttcaat aaacaggcat ttattgctac caaagatata aaagacagac 480 aggccaagat tacaacaaat ttgatggtct acattttttc atgaacaaat gtcattgcat 540 ttagatcagt catcgagcat tgttgtcgag aaatctgtgt gataataaaa catgtgctat 600 t 601 <210> 20 <211> 309 <212> DNA <213> Mytilus galloprovincialis <400> 20 tgttgacatg aatatcaatt atgtggtcat ttttatgaat ttcctgttta caaaactttg 60 aattttttga aaaactaagg attttcttgt cccaggaaca gattacctta gccgtatttg 120 gcacaactta ggaaggagga atttcggatc ctcaatgctc ttcaactttg tacttgtttg 180 gctttataac tattttaata tgagcgtcac tgatgagtct tgtggagacg aaacgcgcgt 240 ctggcgtact aaattataat cctggtactt ttgataacta taaaactgga acaaatgctt 300 cttcatgac 309 <210> 21 <211> 173 <212> DNA <213> Mytilus galloprovincialis <400> 21 acaaattcac aatatttatt tcatccaatt aaaatttatg tgcatgtatc acatatatag 60 caatatttta caagttcctg gtacagttat tcgtcaaaaa atgtcatgaa tgtatcgtac 120 ctcggaaatg gtggtgttga atcgggattt gcttgttagc atacttaaat ttt 173 <210> 22 <211> 406 <212> DNA <213> Mytilus galloprovincialis <400> 22 cccatgcaag acgcgtgcaa taacgccgag gtcggagaca agccctgcct ctgcattgaa 60 acgcccaaga agttcccagg tggcggcccg ccgttcaagt acttttgcgg caactgcacc 120 ccaaagatct ggatgaagga ttgtgaccag tggaaggtcg atggagaata agcgcctcca 180 atggttcagt aactctcact atgataagga aagctcaaga atcgttcccg aaaaaatcag 240 gcagtccatt ggacattctc agccaaaaaa gcaccctgta catttgtcgt tgacattctc 300 aaatttcaaa ttcaacacac aaaattctga aggctgattt taaataggtt gttataacaa 360 tgtttttgga tgaaggtttt aaataaaaca gcagctggcc atcatc 406 <210> 23 <211> 246 <212> DNA <213> Mytilus galloprovincialis <400> 23 gacagacact agagatagaa gaaacgctga tatttggtga cacggaaatt ctttgcaggg 60 caagagatct gcgcaccgga agaatatcag aggtccagtt cgatcttctt taaacattcc 120 gtgttgtgga caataattgg cttattgact gatttgcgga acatgtgatc aaataatgtc 180 atttcatgtt ttatattaca taaaaaaggt ttttactttc aatcaaataa aaagatttac 240 caaata 246 <210> 24 <211> 498 <212> DNA <213> Mytilus galloprovincialis <400> 24 aagaatggaa cgaatacaag aatcattgct attatatttc gaaaggtaaa acaaaaaatt 60 ggtctgaatc agagaggcaa tgccgagata ttggcgggta tcttgtccag gtaacagatt 120 cttccgaaga ttcatggctc gttgatatga tgaaaaaagc aggtcaagat aaaacaaatt 180 actggatggg ggcgacagat tttaaagaag gagaatggag atggaccaat gatctatcaa 240 aagttcaata ttcaaattgg gattctcgtc aaccttcggg aggaacagga aatgattgcg 300 ctcatttttg gcattcatct ggttacaaat gggacgatac atcttgtaat agtataggat 360 tcggatatat ttgtgagaga gctcatggat caaactgtct tccctactcg aaacaattca 420 aacgtaggaa atgataaagc tggaaactgt tcatcaaaca tgtagtctga aaataaaata 480 aaaagtgtta gaaaatgt 498 <210> 25 <211> 545 <212> DNA <213> Mytilus galloprovincialis <400> 25 gcactcagta tggaaaccgg atcacagttt ggaaacaaag atagtgagat gagcaatcgt 60 ccaagggagc agcatgttgg accttcaaac tccggacccg gtcctcattg tcctagatgt 120 ggcaaaactg tgtatgaagc tgaacgagct ataggactaa cagatgcttg gcataaaggt 180 tgcagtaaat gtaaggcgtg taacaagcct gtagattcta caacactatg caggcacgag 240 atagaaatat actgcaaagg atgttatgca aagaactttg gacctcatgg atttggtgtt 300 ggatcattgg ctaaataact gcgaacaaga ctgcctacgt cattctacgt agaaacagta 360 cctgccttgt cgtcacgtga caaaaacaca aacgatcgat caacagctca ctggctagcc 420 gtgcatgctc ttagcttgac cctatttcat gtagaacata caatgtgcaa accacactct 480 taatttatct aaactactat tgtttttaat cttggttata tttttttata ttaaactttt 540 tagtt 545 <210> 26 <211> 313 <212> DNA <213> Mytilus galloprovincialis <400> 26 aacttgtaaa ccagggagtt accaatgtga ttcctgggtt gattgcagtt atggagaaga 60 cgaccttgga tgtccaggtt gtgacagtga tcgtttccta tgtgctaatg atctctactg 120 tattatatgg tggggaatat gcgatggtgt agatgactgt ggtgatggtt cggacgaacg 180 tgggtgctca aatattattt ggagtagcta tttttccagc aagcgcggtt tgaccagttc 240 tagtgctgga aaagtagttt ccagaagtat gaacaaaaga ggcaaagtag cagctgcagg 300 aacaaacatg cac 313 <210> 27 <211> 401 <212> DNA <213> Mytilus galloprovincialis <400> 27 ttacggcgga aagaaaatgt agagaaatag gaggatatat tgtcaaaatt gatgacaaag 60 aagaaaacag caatattgca tcaaacagag cacacagtgg taatcatcac tggattggtt 120 tgacagatct caaggaaggg gtatggcgct ggtcctatga tcagtcgaaa gctgtgttta 180 caacgtggta tcctggtcat ggttccaagg gtacatccag taattgtgtg atgttacatg 240 caagtagaac agattggttt gactacgact gtcattacaa gggtgctaaa tacatttgcg 300 aaagcaattt ctgtttttaa atgatgcaca gaagaaagcc ctcaaagcaa agaaaacaga 360 aaaaaaactc aatttcaaat gttgaataaa agtaaataaa t 401 <210> 28 <211> 580 <212> DNA <213> Mytilus galloprovincialis <400> 28 gcagtaggct tggggtcagg ggccttatgt cgctacagag tgtgtggcac gaagacggtg 60 tatttgtatc cacagtgctg ccccactact ctgcatcagg taggggtacg ggtttgttac 120 aatacctatc gaatctgcga ccgtaaacga tcgattggca gccaaaacga catcgaagtt 180 ggttttccct gcgattttaa gaagtatgat accgacaaag atggatcaat cagcctaaaa 240 gagttcaagg ctgccacgga attaaacgag agtgcggaga ccattttcaa tggtgcagac 300 aagaacggcg acggtggaat cgattgtaaa gagttcaaga aatcaaattg ggagtttgaa 360 tgcatacctt tcggatgcgg cgacgaaact caagatgagc tttggaatgt cgagtaacta 420 acattcagca tttttcattt atttcatatt gactaggact ataccactct ttacattaaa 480 tgataagtaa cgtaatttac ttctttttgt catcgttttc acattatcaa gcattcactt 540 gttgtaatac gtaatgttct aataaaagct gcacaaacat 580 <210> 29 <211> 222 <212> DNA <213> Mytilus galloprovincialis <400> 29 acagtcggca caggtacaca cacgggaaaa acgatattga aaaaacccca accagacggg 60 acaaacgatg ggaaaaacag taaaagcgga cacggccaag gctggtgtcg agggaggatt 120 aacatttgat gaagtgtaga gcgtagtatg cccattccgg cgtgcatggc tccgactttt 180 acctcccacg gcacctcggc ttttcggtcg ataccgatac gg 222 <210> 30 <211> 296 <212> DNA <213> Mytilus galloprovincialis <400> 30 tttgttatac cagcggcact tggagcggca ggttttactg ctacaggaat tgcagcaggc 60 tcattgggag cagctgcgat gtcagcctct gcacctgtcg ccaccgggag cgttgttgca 120 attgctcagt ccataggggc aacgggggct attggattga cagggaaggc catcggtggt 180 ggggcagctt acgctgccta tcgcaccaca agggatcgtg attaactttt attgtacttt 240 ccaatgaacc agtttatttt attgatttgt atataaagtg tagttaccac tcattc 296 <210> 31 <211> 188 <212> DNA <213> Mytilus galloprovincialis <400> 31 tggtgacaca tctgaacatt gatcgctgtt tatttcggcg gatctgtttg gtttggtgtc 60 cctctttaga acacattccg cattatatgg aactcccgct gcctcgtcgc acggttgaga 120 tctgtttaaa ttggcctctc gtttaggccc tgggtataaa aagttaacat ttgacttcta 180 aagtttct 188 <210> 32 <211> 105 <212> DNA <213> Mytilus galloprovincialis <400> 32 gagtgaatgc ttaatcagcc gacataaaca atgatggtag aaaaaaaatg aacaaattta 60 aatgtgtgct tttgctaatt aaataaataa aatttcaact acttt 105 <210> 33 <211> 555 <212> DNA <213> Mytilus galloprovincialis <400> 33 tacatcttca ggagctattg ctggagctgt tgctactaaa acagcggcaa gtgctttcac 60 aggtgctctt ataggaggct tgtctgccgg aagtgttggg tctgtaatgg ctggaagtat 120 tgcgttagca tctgggacac tgtcatcaag ccctttaggt ttgttgaccg ttggaaccaa 180 ttcaaatgga aatcagataa cacacgattg ttggaaacaa gttgtacacg aagcatcaca 240 ggagccgtcc aatgggataa ttctgaaaga gttgatctct catccgaatg ttgctgacgt 300 aacagtaata ccatgtggta atcttccata tgtcgtcatt gagaataggt ggaatgaaaa 360 atttgaactc gaatatgttg cactacttga atctaacaaa ttgtttttac acgttcagcc 420 actctgaaac tgaaaaaagc cactacaaca cttataataa tatgtccgat gtaaacttta 480 atattgtaca gtagtataac atgtggacat attgtaaggt attaatttaa gataaagtaa 540 acggtgtgaa taacg 555 <210> 34 <211> 230 <212> DNA <213> Mytilus galloprovincialis <400> 34 ctttgggtca ctcccggagc cacgcctgtc tgagggtcgg tgaaacatca atcgcaccaa 60 acgggttcac gcccgctttg actgcgcctt gggcttttgt cgcagcggac cgtttttaac 120 ggtacgcttc gtcgccttaa atgcagaccc atgtcgtctc gctttgcctt tcggtacttg 180 tattcttaat ttctccgccg ccgtacggct gtggagggga cgcacgcctg 230 <210> 35 <211> 776 <212> DNA <213> Mytilus galloprovincialis <400> 35 ttatctaaaa ataagcttat gatcgggggg tcagtaatta tgataaaaac gagtaaaaat 60 attatttttt tggtatcact gttttgtttt accttaaaag attaacggct aaataaaggg 120 ttttaaatga actcggcaaa ctaaacttct cgactgttta acaaaaacat ttccttttga 180 tgtaagtaaa aggtagtccc tgcccagtgc aactagagta atgtctgttg taaacggcgg 240 cgttaacgtg agcgtcctaa ggtagcgcga taatttgctt ttcaattgaa ggatggtatg 300 aaagggttaa cgaagaaggt gctgtgtcta aaaattcaat ttaaactaac tttaaggtga 360 agaggccttt atgtaaaaga aggacgacaa gaccctatga agctttatct taattggagc 420 tttcaggctc ttatacgatt ttgatgggag atcagtagaa ataagtcttc tactatcata 480 ttaatcttac tagtatttcc taactttata tgtgtggcta gctactctag ggataacagc 540 gcaatttctc ccgaaagatg gtattggagg ggaagattgc gacctcgatg ttggctttag 600 atatcctaga ggcgtagaag cttctgatgg tgggtctgtt cgccctttaa aatctaacat 660 gagctgagtt cagaacggcg tgagctagtt cagtttctat cctctttttg aaatgagcta 720 attttgtacg aaaggacttt ttcgctaaag caatgctttg tcacaaatct gtgttt 776 <210> 36 <211> 500 <212> DNA <213> Mytilus galloprovincialis <400> 36 tgtatacagg acaagttgga ggtaactatc atagtaacaa aggaggaggt tccaaccggc 60 tctgtttacc caatgaccct gaaaatggta aagcatactc ctatagtaat gatggactat 120 atggtgctga gtaccagata aaatccagca ataatccatc cgggtttcct gcaagtttgt 180 ataacaaaga ggttccatgt tctgtttgca ggcggaaggg gaaagtttct gttttaatga 240 tagcaggaag aaagtcctgt tacaaaggat ggaaatctgt gtacagtggt ttcttgatga 300 gtgaacacac gacttataac tctaaagact ttatctgtat ggacggagac gcagaaccat 360 tggacaatcg gtcctccaac gaagaaggtg ccttgtttta tcctgtcaga gcaaaatgtg 420 gtagcctgag atgtccccca tacaaagata acactgaagt tctttgtgcc gtttgtacaa 480 aataaatatg aaataatggt 500 <210> 37 <211> 451 <212> DNA <213> Mytilus galloprovincialis <400> 37 cagggtcagg atcgttttcc tatgacatgg acagacaagg tttgaggaga ctttacaaca 60 gcccagttac gaaagttgaa agttactcca gacccctttt aaagattggg agtgctcaaa 120 taccccatac tggggttgta gctacaacta aagatggcgg caggtttctg gtacacaaag 180 gatatggtta tgggaaatcg tccagtacag tagttgttga tgctaagcat atgtcaaaca 240 gatggtcaaa gactggagaa aaatatacaa gtggaaaaac aatatctgat atcatgaaag 300 atggttatgt cgatcgaaac tatggcagta aaatatgctg gcaatcttct caaaacataa 360 tggacaaggc aggtcgcaga aaaaggcgag cctgtcgtta aacagtaaac cattctaacg 420 tatatattaa ataaatatat ttcaaaatgt t 451 <210> 38 <211> 781 <212> DNA <213> Mytilus galloprovincialis <400> 38 agatgtagcc ccctcttcag tcgatgatga gaacgaggtc gaggctccga agccagatgt 60 taccccctct tcagtcgatg acatggagga cgatttcgag gctccgaagc cagatgtaac 120 cccctcttta gtcgatgatg agaacgaggt cgaggctccg aagccagata ttaccccctc 180 ttcagtcgat gatatggagg acgatgtcga gactctgaac ccagatggtg ccaactttga 240 gtcaaaaggt gaaacggaga atgagtttca ggctctgaaa tcagaagtat ctatgttgga 300 gacagaaatg aaagaagtcc gacgtcgtca ttatcaccgt ggcaatggtt tccgacgtca 360 ccgtcgtcat catcaccgtc gtcattatca ccgtcgccat ggtcgcgtat ctggaactgc 420 acagcttggg tattcgagta gacgaaacaa acaacgagtt atgtacatac gatctcgtag 480 acctttccac aggacaccag ctttagtggt tggaatgacc tcactggata cctacagggg 540 aacgaatgtg aggataacta cgaatgtact ccatctttcc aatcatggat tcattttgca 600 tattaaatcc tgggccggct cgattaccta caatgttgtt tacacttgga tggcgtgtcc 660 caagtagact ttcgcataat catagatgtg cctattcctc tatgagtgta ttccaattgt 720 aactttctac aaacccactg tttcattctt gctatttcta tagaataata aagcttcaaa 780 c 781 <210> 39 <211> 684 <212> DNA <213> Mytilus galloprovincialis <400> 39 agatgtagcc ccctcttcag tcgatgatga gaacgaggtc gaggctccga agccagatgt 60 taccccctct tctgaatcag acgatgatat ggaggacgat gtcgaggctc tgaacccaga 120 tggtgccaac tttgagtcaa aaggtgaaac ggagaatgag tttcaggctc tgaaatcaga 180 agtatctatg ttggagacag aaatgaaaga agtccgacgt cgtcattatc accctcgcaa 240 tgctttccga cgtcaccgtc gtcatcatca tcgtcgtcat tatcaccgtc gccatggttg 300 cgtatctgga actgcacagc ttgggtattc gggtagacga aacaaacagc gagttatgta 360 catacgattt cgtagacctt tccgcaggac acctgcttta gtggttggaa tgacctcact 420 ggatacctac aggagaaaga atgtgaggat aactacgaat gtactccatc tttccaacca 480 tggattcatt ttgcatatta aaacctgggg cggctcgatt acctacaatg ttgtttacac 540 ttggatggct tgtcccaagt agatgttcac ataatcatag ttgtacctat tcctctatga 600 gtgttttcca attgtaactt tctacaaacc ctctgtttcg ttcttgttat ttctttagaa 660 taataaagct tcaaacgaaa atat 684 <210> 40 <211> 529 <212> DNA <213> Mytilus galloprovincialis <400> 40 tatgtcaatg aaggaggttg atgaacaaat gttgaacgta cagaacaaaa acagcagcta 60 cttcgttgaa tggatcccca acaacgtcaa gacagccgtc tgtgacattc caccacgtgg 120 tcttaagatg tctgccacat ttattggaaa cagcacagcc atccaggaac tcttcaagag 180 aatctcagaa caattcaccg ctatgttcag gcgtaaagct ttcttgcatt ggtacactgg 240 tgagggtatg gacgagatgg aattcacaga agccgaatcc aacatgaacg atttggtctc 300 agaataccaa caataccagg atgcaacagc cgaagaggaa ggagaattcg aagaggaaga 360 aggagaagag gaagcttaaa catttcctaa agctcaatga actttaaaaa agaataattt 420 taaaagtgaa actaaagaat aacttactga tatactggat acaaacaaac aactgtccag 480 ttatctattg aaatattaca ccatcatgat attaaattat tgaacaatg 529 <210> 41 <211> 178 <212> DNA <213> Mytilus galloprovincialis <400> 41 acgaagacag aaatgtgtat tcctgcacgg ccaccaagac acaacggtgg ttaaaatcaa 60 tcctacatcg ctttttttat aaaatcttat ttatattgtt atatagtgtg cgtatttttt 120 taaatcattt aataatcatt taatatattg aaaaagtgag ggacaacccg cgccccca 178 <210> 42 <211> 344 <212> DNA <213> Mytilus galloprovincialis <400> 42 tatctgttgt agactgttct tgctgcaggg aagttgtaat atctgatcaa agatgaggat 60 tcttttaatt attgttgtga tttttgcttt cttggctttg attgatgctc aggcaaaagt 120 gaaggctaaa acatgcatta ccaaatgtac aaagaacatg cttggctact tccagtcatg 180 cgattcttgt gaagcctacg tcacctgtgt tttcagagta aaatatgatc ggaaatgtcc 240 agggacactt aagtggaacg acaaaaccaa ggaatgcgac ttcataacac caacctgcga 300 aaagatttga ttgacattta caataaagca aaattcttag aaaa 344 <210> 43 <211> 239 <212> DNA <213> Mytilus galloprovincialis <400> 43 ctggtgtgta ttttccaagt gtgcgtttcg ttcatcagaa tttaatgctg ccctcggtgc 60 atcagaaaat taattgagac ctacggggag atttgatgaa atatcgaaat ataactaatg 120 taaataacta tacaatagta acagaatgac aattgcgttg tgttggtatg ccaaggtgtg 180 gttatgcaag tttgtttact ttatgtactt taagtttata attaaaaaaa atgaaatat 239 <210> 44 <211> 471 <212> DNA <213> Mytilus galloprovincialis <400> 44 tcaaggaaca ctacaaggga ctaattgtaa cagtgatttt tatcaacctt tcaaattcct 60 gtcatctgga aatagtcgat gtgtatttga aaaatcatac tgcaatgagg aaggacaagt 120 tattcaaagt aatggtacta ccagaaatga cagatcatgt cgttgcgatt atacgagagg 180 atatgatttt aaaacacgac caaatcatcc atgtttgtgt ttaccatcaa aagaagattg 240 ttcctgctat cgtaggaaat gcccgaccaa acacatttta tcaccagatt atgagtgcct 300 gaatgaaaac gagtggaaaa ttggctttac ttgtgactta atatcaacag ttgtgacgtc 360 taaagattca aaaatatcat tatcgacaga caattcatcg ttggactatc atgtaatagg 420 tatgaacata agttatataa ctttagaatt cctaattgat acagggtcta g 471 <210> 45 <211> 219 <212> DNA <213> Mytilus galloprovincialis <400> 45 caccacaggg ggaaagaacc gtaaagtggg tgttaaaatg atttttggga gaaccgagct 60 gcaggttcgt gctgtaaaca aaacaacagg agaggaaaca aaggctgaat ttgacttttt 120 gcgatgaatt tcattaaaca tttgaacgga ttatatttac atttattgtt ttggtagaaa 180 caaaattgta gaattattac tattaaaaaa gtaaatctt 219 <210> 46 <211> 327 <212> DNA <213> Mytilus galloprovincialis <400> 46 acctagatca ggagtatata atgtaatttc caatatgttt tgtccacgag gaacgcatca 60 tcttggtgac tgtttttatg gtcctaccca cggagacgga aaccattttt ataatccatg 120 ggagcctagt atttgtaaag ctactggtga ataccgtgct cagcgtattg ttgtgggtgg 180 tgtttcgtgc tcttgataac attgaaacgg acatttcatt ctcttcgata attattgtcg 240 caatgcatgt gaactgtttt tgcacaatgt cggaatttgc attttttgtt taattctaat 300 ttgcatatta aaaataactt cataatt 327 <210> 47 <211> 129 <212> DNA <213> Mytilus galloprovincialis <400> 47 caaaggataa agtttacata aaaactcatg ggacacagaa atatatgtat ggaaatgttt 60 ggtctacatt cacaggtttc aaaatctaag ttttaaaatt gtttaataaa gatactggaa 120 gtgcaattc 129 <210> 48 <211> 152 <212> DNA <213> Mytilus galloprovincialis <400> 48 caatcccaac gacctaaaaa tattctttct cacctgtttc tcacgaacct acaaaaaaaa 60 actgttgtca aaatttaaaa atgttgtaaa attttcttcg tactcttaaa tgtatgtaat 120 tttctatctt taataaactt cttgcatatt ta 152 <210> 49 <211> 423 <212> DNA <213> Mytilus galloprovincialis <400> 49 gaaaaagggg agcttggaaa caactgtgca gatgccggtg gtcacttcaa cccatttgag 60 aaaacacatg gacctccgaa cagctacaac aggcatgtgg gagacctggg taacatcgta 120 acagaagacg ataacaaacc aacaaagttt ttgatatatg atggaacaat taccctgaaa 180 agctattata aatcaagcat tgtaggtaga gcaatagtga ttcatagtgg tgaagatgac 240 ttaggacagg gaggagatga gggaagtctg aaaactggga atgctgggaa gaggctggcc 300 tgtggcatta ttaaattgca ataccggtat ccatattggg gttaactaac aaccaataat 360 aaatcacttg ttaaaatttg attgctattt gacactaaaa atatatgaga tatcatttga 420 ttt 423 <210> 50 <211> 164 <212> DNA <213> Mytilus galloprovincialis <400> 50 agagtgacaa tttattaaag aaaatgggtc tttacttttt cagtatcatc aagctgctga 60 taacaaacgt ctttttgcct ttcttaaaaa cagaaattgc tctcacagat gtaacggtag 120 gttgttgaac aggtgtaatc gaaaggtttt agtttattga catg 164 <210> 51 <211> 500 <212> DNA <213> Mytilus galloprovincialis <400> 51 aaggtcaagg aggaatcgat gtggtacctg gtttaggcac aggccatgca ctgctgctaa 60 gtgtagtgat ccttcaagag ggaaaccatt ttactttgga acaactcgtt tgttttgtat 120 ggcaaaagga aaacgtggat cgtcaccatc tcgaccaaca tgggcactaa agcatagatg 180 gatcgaatac aatggccatt ttttcgaacg attgaagaat ggaggagata tttttagctc 240 aaatgcccct aaggactcca ataaatgttc ttctagtcgg gaaagtcatc cagccgggta 300 cagcagtttg agcatagatt gtatgaaacg ttgtacgaat aagtacaggc aacgctatgg 360 tggataccgc ctgttgtcaa ataactgcca ccactttgct aatcgcttct ccgatatatt 420 atgcagcagt tccacgtgtc cttcgtggtg tgcttgaatt gatataatct tagtttacta 480 tcattaaatg taatttcctc 500 <210> 52 <211> 500 <212> DNA <213> Mytilus galloprovincialis <400> 52 aaggtcaagg aggaatcgat gtggtacctg gtttaggcac aggccatgca ctgctgctaa 60 gtgtagtgat ccttcaagag ggaaaccatt ttactttgga acaactcgtt tgttttgtat 120 ggcaaaagga aaacgtggat cgtcaccatc tcgaccaaca tgggcactaa agcatagatg 180 gatcgaatac aatggccatt ttttcgaacg attgaagaat ggaggagata tttttagctc 240 aaatgcccct aaggactcca ataaatgttc ttctagtcgg gaaagtcgtc cagccgggta 300 cagcagtttg agcatagatt gtatgaaacg ttgtacgaat aagtacaggc aacgctatgg 360 tggataccgc ctgttgtcaa ataactgcca ccactttgct aatcgcttct ccgatatatt 420 atgcagcagt tccacgtgtc cttcgtggtg tgcttgaatt gatataatct tagtttacta 480 tcattaaatg taatttcctc 500 <210> 53 <211> 445 <212> DNA <213> Mytilus galloprovincialis <400> 53 cataattgtc aaaaacagga tttgcggctt cgattataca ctaaaacgtc gttgatatgt 60 atgagagaag aacagatgta ttggattgga aatcacatcg cagagaagat tatctgggca 120 aatgaatcct tgccagaaga tgcgttatgc atcaagttag taataactgc atccggttcc 180 agtcttgaaa ctgaaagatg caacactaaa ctgcaatcat tgtgctatac gtcacagcaa 240 acatctagtg ggaggattga tgaggcttac atgacatcag catcgacaca atcaaaaggt 300 tcaacaggtt gtacaactct aaagggtcca gtaaacgcag ggtctgattc agatggggat 360 aaaccagatt tgattccaat tgtagccggg agtgttggag gatttgtagc aatcgtgatt 420 gtcgtaataa ttgtgttcat cattc 445 <210> 54 <211> 197 <212> DNA <213> Mytilus galloprovincialis <400> 54 gaaaaacgat attgaaaaaa ccccaaccag acgggacaaa cgatgggaaa aacagtaaaa 60 gcggacacgg ccaaggctgg tgtcgaggga ggattaacat ttgatgaagt gtagagcgta 120 gtatgcccat tccggcgtgc atggctccga cttttacctc ccacggcacc tcggcttttc 180 ggtcgatacc gatacgg 197 <210> 55 <211> 269 <212> DNA <213> Mytilus galloprovincialis <400> 55 cggaagaaag gctgtcgtcg accaaaccgg gggtcccccc ggcacagtcg gcacaggtac 60 acacacggga aaaacgatat tgaaaaaaaa aaccccaacc agacgggaca aacgatggga 120 aaaacagtaa aagcggacac ggccaaggct ggtgtcgagg gaggattaac atttgatgaa 180 gtgtagagcg tagtatgccc attccggcgt gcgtggctcc gacttttacc tcccacggca 240 cctcggcttt tcggtcgata ccgatacga 269 <210> 56 <211> 290 <212> DNA <213> Mytilus galloprovincialis <400> 56 gataatcgtc tacgaattac aatacaaacc gacggcaaag tcactgcaag gtggacagat 60 gaaacatggg gaaagatatt caacgatgtt ggaaccgtca tcaacaattt aatgaaaagt 120 gtcattaaat ccattaaagg aactagcgta atcggtgacg tggaagccat cgatgcgatt 180 aaaaaaggcg ctattaaaaa ttgattgcat ctgcagaaag gttcttgttt cgctggatct 240 aaaatgtact gttttttttt aaacttaaat atatattaaa atatctagac 290 <210> 57 <211> 252 <212> DNA <213> Mytilus galloprovincialis <400> 57 ggagattata atggttattt ttgtccctgt catggttttc attatgattt atttggaaga 60 attaggaaag gtccagctcc tttgaacctt gacatccctg accatttgtt tccagaagga 120 acaatgctgg ttattggtta accaaggatt ttaatagaac aatgatttta tttattttgg 180 tgaaaatatg tacaatggta ccagtggatg atagtttata tgttcacata ttgtaaagaa 240 atttgacaag tt 252 <210> 58 <211> 145 <212> DNA <213> Mytilus galloprovincialis <400> 58 gcagaatcag caagtatacg gagatttaat tttgattaga tagaaaaatt tataaaacgt 60 caagtcaact aaacaattcc tttattaagt tttctatgac atatttgtgc cttatgttta 120 agatataaaa gcttcaacat gaacc 145 <210> 59 <211> 353 <212> DNA <213> Mytilus galloprovincialis <400> 59 tttggacaga gtttaaagga gctgagaatt catttatgcc agaaatctat agcaagtcag 60 ggtgtcagag agttcataga gaattactat gttggtgtta agcagagtaa ccctaaattt 120 cctattttaa ttagagaatg tagtggagta caaccaaaag tctatgctag atatgaattt 180 ggaaaagaac acagcctacc attatcaaac atgacaaaag accaagtgtt ggatgctttg 240 aaaacattat cacagagatc ttgatattta tatcaagctg gttatttgtg aatgactagt 300 attgttcata catgtgattt aaaggactgt aaaataaatc ttaaactaca aaa 353 <210> 60 <211> 333 <212> DNA <213> Mytilus galloprovincialis <400> 60 tttgatgagg cttgtcccga caggttgtcc gttaatcctt gtgcctttta tagtagtagt 60 tgagctaatt agtgggatac ttcgcccttt aactctagtc ttgcggctaa cgttgaattt 120 aggggctggt aaagtaatct taactatatg tagaagggag ttggtagtcg gttggttaaa 180 tagaagaagg ttaattacgg gagttggggg tattaaaggc ttgctaatag gtggaggtgt 240 ctttggagct gctgaagttg cgattgcttg tattcagtgt tacattttct gtgtattatt 300 gtgtctctat actgaggacc ataggagtta gat 333 <210> 61 <211> 252 <212> DNA <213> Mytilus galloprovincialis <400> 61 aaagaaggat cgcctggtcc tgcttttgga atatgtatat tagctggagt tctctccatt 60 gtagcggcag ttattacagg ggtcgccact gtaaagaaag gagatgatta ttatgattaa 120 acattttctt aagaaaggag atattatttt atacaattgt taatttcgta gtgtattttt 180 tttataacat ttctatgatc tacatgtata tataaaattt aggactaaaa taaataaaaa 240 tatgacaaat tc 252 <210> 62 <211> 224 <212> DNA <213> Mytilus galloprovincialis <400> 62 ccaaagtgtg ttatcgcctc tgtttaaaaa aaacctaggt tggggctatc aaccaagcgc 60 ctacaggcag gaggctatat agtaattccc actgtattct tgctcaaaat ttttttctat 120 ggggagtacc agatttttcc cttatagggt taagaaaaag gatgagaccc gtggcatcat 180 ttcaacctct tccccccccc ccccctcctg ctcccctttt tcgt 224 <210> 63 <211> 233 <212> DNA <213> Mytilus galloprovincialis <400> 63 ttcttgtgta tacttaataa ttaataattt aggatcaaaa tgaatcttat gtttctaata 60 cttaaataat ttcatttttg attagaaaca ttgaattaat gtaattgtaa tatgaatacc 120 tatagtacta aatctggcat aatttggtgc attatttacc gattttagtg tcgagttaaa 180 ataaatatgt ttcaatcata acatattaca taattttcat taaagttgca ttg 233 <210> 64 <211> 143 <212> DNA <213> Mytilus galloprovincialis <400> 64 gatggaacga atgcttcccc gcaagactgc atgttttttt atgcaggaga aagatatgca 60 attcacaatc taccgtgtga tcatcctaga tggagtagtg gttatatttg tgagaaatga 120 aaataataaa aaatcctttg atg 143 <210> 65 <211> 237 <212> DNA <213> Mytilus galloprovincialis <400> 65 tgtgctaaga catttaaagg tgcttggtgg tacaataaat gtcattcttc taacttgaat 60 ggactatact ggggaggagc tcatacagag tatgcatctg gtatcaattg gggatcctat 120 ggctaccatt attcgcttca agctacaacg atgatgattc gtgcaacatg aacttgagct 180 catatgttat ttataaaata cacgcctgta tcagtatgta ataaaggttt gaagacg 237 <210> 66 <211> 239 <212> DNA <213> Mytilus galloprovincialis <400> 66 caaatttacc aagcaatcta cggaagaagc actggcagaa gtgtttgtcc acacccctct 60 atcaggactc ataattgtag atcttcagta tcgaacagaa tagtaaaatg gcagtgcaat 120 gggagaagtc tgtgtaaatt agaatctaat aatcaccttt ttggagctat atgctatggc 180 acctacaggt acttggaagt aaaatacaaa tgtaaataaa taaagtatgt caaaatgtt 239 <210> 67 <211> 217 <212> DNA <213> Mytilus galloprovincialis <400> 67 gaaagtattt ggtgatgact gtgcaatatg cttagatgag aagaattcag cgatgacgac 60 cattgttggt ctttcgtgca agcacacatt tcacaaatcg tgcgttaaag aatggatgat 120 acataaaagt cctctcactt gtccggaatg tcgacagcca tcaaagaaat tttcttcaaa 180 aacaatgatt tcggacttaa tatctacact gacagct 217 <210> 68 <211> 580 <212> DNA <213> Mytilus galloprovincialis <400> 68 cgtatcattg cattcactgc agagttgtcg atagatacga aactagggac gaaccaggct 60 gtggtatata acgaagtttt gcttaacaaa ggagatgctt acaatcgctt tcatggtcat 120 ttcactgcac ctgtaagagg aatctatcta ttatcagcca gtatgcttgt taaacaagtt 180 ggaaagcatg tgttattaga tatggtccac aatggtgtca caattgctgt tttaagatcg 240 ggaagctatt ataacacaca gtcgtctagg agtttcccag ttatgttaca acgaggtgat 300 caagtatgga tgcgaacatc ttcagttgca gcatcttccg ttggttacac tttaatagga 360 aaagctcatt ctatgtataa ctcattctct ggtgttctct tgaattagtt ttgatggttt 420 tgtgctgctt tactcacatt atgtgaaatg aaattagtat acattacttc ttctattaca 480 accattactt ttactactgt aactaatatt actgccgcta ctaataataa agagattcaa 540 gtactagtat actgaaataa acttgatttt tttttaattc 580 <210> 69 <211> 401 <212> DNA <213> Mytilus galloprovincialis <400> 69 aaagctgcat atgtttatta tagatagatg acattagttt taaaataatc aagagtaaag 60 gtaatgttca aagtattgtt aaagaaagaa taaattttgt cttcggggta ggatatgctt 120 tttctcaaca aattatcaaa taaatggcag ggaaattttt acttttcttc attaaacaca 180 ccaagatgga caagttctac tgttacacag gatttcggac atgcggtttg catatatatg 240 gcagttgttt ccccaccatc catatgatcc atattttctt ctatagttct ttgtacagcg 300 tttgatacaa tccacgctta aagaactata tccagctggt gtacgttcac gatgggttga 360 gcatttgcca gactcttgtg aaaatccttt agtaaaaaaa t 401 <210> 70 <211> 118 <212> DNA <213> Mytilus galloprovincialis <400> 70 actttttcta ataaaaattt attgaggaag ttctcatatc ttatagtttt tccaaatatg 60 aactatttga tcaaaatgct tttgatgtaa tctattacaa tatccttttt cagcatat 118 <210> 71 <211> 413 <212> DNA <213> Mytilus galloprovincialis <400> 71 taggaaagga atgtcaagaa tatgttgata ttgtggcgga tgtggcatct aacgatgatg 60 cgtattacat gttagttgca ttgattccat gtgcacgttt gtggccatgg cttggacagc 120 aattgactgc aaaacagaat aattttgggg catacactga ctgggtcaat tcaaatttcg 180 atccatctta taaaggatat aagaagttgg aagtccgtgt gaatgctgct ttctcaaata 240 agcaaatcga tagaaacaaa gctctgagta tctactcaaa atgcatgaat ggagaagctg 300 gcttttttgg ttcagtaccg atatgaaaac attcagcttg ttttgtactc attatgtgac 360 gaaaaagaaa ccaaaaaaat tgtaaattga ataaaataat aaagttgaaa tgt 413 <210> 72 <211> 294 <212> DNA <213> Mytilus galloprovincialis <400> 72 attgaaatat tgctccacca agctcattac tgaatcctgc agacttgcgt gtggtttttg 60 cacggcgcga aagcgaagct tgccggaaga gagaggcatt gtaactactg tcggaacgta 120 ttgccccact gtagatgcgt gtttatgcaa ttcagcaact gtacgatcca tctgcgcatc 180 aagatgcgtc caagtccaag ctggttaacg atgacgcaag aaaaactttg tatcttcatt 240 taatcggatt gaataatcgg catattttaa ataaattgac tacaaaagca acgt 294 <210> 73 <211> 118 <212> DNA <213> Mytilus galloprovincialis <400> 73 actttttcta ataaaaattt attgaggaag ttctcatatc ttatagtttt tccaaatatg 60 aactatttga tcaaaatgct tttgatgtaa tctattacaa tatccttttt cagcatat 118 <210> 74 <211> 321 <212> DNA <213> Mytilus galloprovincialis <400> 74 aaagaccaga gaatgggtat gagtatgtaa aatttcacac tggaggaaaa gagaaattga 60 atcgacataa actagaacag ttaagggcgc ttgtcagctt tgcaacatgt gtgccactgg 120 actttgtggt tgtgagcgga atagaagcta caaacagtat cctagttacc ttcatgatcc 180 cagagggata tgctcatgtt ttatccgaac tcaacaacaa agagaaagaa tacttaggtt 240 cccagggggt tgatgcaata tggtacaatg gacagctgat cagttgcata gatatcgaaa 300 tcggggaagt tgactctata c 321 <210> 75 <211> 216 <212> DNA <213> Mytilus galloprovincialis <400> 75 aatggagtta ttaaggacat tacgtgggct gattcgatgg atcatccaga ccgtgccgta 60 gcgacaactg ttactcccct agatttgagg aaaggtgata aagtaaatgc cagaataggt 120 cctgcaaaag gtggagcaat tctggaaagt acgaaccata tccgttgtag cttctctggt 180 tttaaagttc agtaataata aatgttttag atcttg 216 <210> 76 <211> 365 <212> DNA <213> Mytilus galloprovincialis <400> 76 attttaattt ttaagtctgc agcaacaaaa tttacacgtg gagagcttca agatgctaga 60 agtgactcag aagatgatga aaaggatgag gatgatgatg atgtacttaa tgatgaaaac 120 ttcaaagaaa tgtccaacaa taatacagga aacgatggca ttggtcaata aaatgtgcaa 180 ttttacctca aaaggcagcc tttacaaaac atgattttgt tctacttaat atatattttg 240 tgttgtgaaa gtaaacttaa tacaatcgtc agatttatga tatatgtttt atatgtttta 300 tatgctttat atgcttaata tcctacaaat atgcttccta ttaaaatgta tgtaaaataa 360 atgtg 365 <210> 77 <211> 172 <212> DNA <213> Mytilus galloprovincialis <400> 77 caggtaggaa agtttaattt aaaatatttt aatttatcaa attgctgaaa tagtaaatgt 60 ctcccacagg tttatataac ccgattcgtg aaaatatcat tgttatttcc atcccttata 120 ttcctgattg tttggattca attggttgga gtggttgtcc aggtctggct ta 172 <210> 78 <211> 544 <212> DNA <213> Mytilus galloprovincialis <400> 78 ggaagtgatt ataatactag aggaggtggt gtgaattatt tatgtctccc aaatgatcca 60 gagaatggac aacaccaatc atataacaat atccaggtat atggtagtga atatcgactt 120 agttcatcag tcaagccgtc aggatggtat gaatctatgg ccaacatgga agtgccatgt 180 tctgtttgtt tcataaaacg cagatcaacg gttttgatga ttccaggtcg gaagacttgc 240 tataaaggat ggaaatcaga atataatggt tatctgatga gtgatcattc aactcattat 300 agaagggact acgcctgtgt tgacaaaaaa gcaagtccat tggactataa gaaagtaaat 360 gaacatggcg cttattttta tccactccga acccaatgtg gtagtttgag atgtcctcca 420 tataccaatg cagcggatgt gttatgcgtc gtttgttcaa aataaaaaca ttatgagtta 480 cattgataac acgaccattt atttgtgttt atctataaat tgattaaacg actatatata 540 tttg 544 <210> 79 <211> 192 <212> DNA <213> Mytilus galloprovincialis <400> 79 caaggctttg gacacatggc ggaggtagag gcgaagtagc atctcatacg ctaaatcttc 60 cactcaaaag gggagaccag atgtgggcca gtggatctcc cgggaaaaag atatacgtag 120 gagagcctta taacgtattt tcaggaatat tgattgcaag tggaaaattc aattaaacat 180 acatagatca at 192 <210> 80 <211> 401 <212> DNA <213> Mytilus galloprovincialis <400> 80 atttttctac taaaggattt tcacaagagt ctggcaaatg ctcaacccat cgtgaacgta 60 caccagctgg atatagttct ctaagcgtgg attgtatcaa acgctgtaca aagaactata 120 gaagaaaata tggatcatat ggatggtggg gaaacaactg ccatatatat gcaaaccgca 180 tgtccgaaat cctgtgtaac agtagaactt gtccatcttg gtgtgtctaa tgaagaaaag 240 taaaaatttc cctgccattt atttgataat ttgttgagaa aaagcatatc ctaccccgaa 300 gacaaaattt attctttctt taacaatact ttgaacatta cttttactct tgattatttt 360 aaaactaatg tcatctatct ataataaaca tatgcagctt t 401 <210> 81 <211> 438 <212> DNA <213> Mytilus galloprovincialis <400> 81 ttgcagtact tcttatagtg caagacgcag atgcaagctg tgcttccaga tgtaaatctc 60 gttgtagagc cagacgctgt agatattacg tgtcagtcag atatggatgg ttttgctatt 120 gcagatgtct ccgttgttcc agcgagcata ccatgaaatt ctcccctgaa agtgaaggac 180 cagctgagat gccagcacag atgaatgacc atgagcaatt ccaggacatg cagaaaggag 240 aaaccgaaca aggtgaaact ggaatgtaaa gagatggcct tataaagtga cgttgataca 300 cattctgttt cggagtggta ttctgattcg aactgtgtta ataggccttt ccctttcgtt 360 tcacttgtat aaaatgtctt agatttctgt aattgttatc taatatcata aattctaaat 420 aaaattgtgt ttcaactc 438 <210> 82 <211> 232 <212> DNA <213> Mytilus galloprovincialis <400> 82 tgaccaacca atcgtaacac aaatggtcga aagacctacc gtagtacgag agagacatgc 60 ttatcctaca gtaactcgcg ttgtggaccg accgaacgtt cacggtagac caataagtgg 120 atctcaaatt gtacgagggt caatgccgga aggagaagtc attgaacgtg tgatacaaat 180 gccatcacac ggtcctccat cgtacgaata tagcaagcaa gttattgata ct 232 <210> 83 <211> 784 <212> DNA <213> Mytilus galloprovincialis <400> 83 gacgttggcc aactggaagc ctctcgttac caaaaccaaa atctggatgc ccttctggct 60 gggctcaggg atataggtac caggacaatg aaaacaacag taataataat cgtgttaccc 120 taggttcgac ccatcacttt tatggttatt ttgggtcaaa cacaaagatg tactactgta 180 ctaaaacaac ctacagcgga tcatcagatt cctggccaaa gggaaattat tgcttcgcaa 240 gaaaaggtgg ttcctgtcct tcaggatttt gcactggatt tatctactgg gacgacgaag 300 atacttcaaa tagtaactac aaaggtggtg ttcttccaga tgggcaatac gacaggaaca 360 caaaaataaa ctactgttgc aggtctgatg gctctgccta caggaacatt aacctaccaa 420 caagtaagcc tttctatctg tacaagtata catcgatatg ccagcgtgta cgtggaatgg 480 ctgtccgtca agaatcggtg cagatggatg acgaagatgt caggaacgcc agtagtgatg 540 gtggatgtca tccaagtaaa tctggctcaa ccaaagtgga cttctgttat tattattagt 600 gataacattt ctcctgtgta gatacttttc agacaatcat cttttacctg aatgactctt 660 ttttattatt gctttacttc attaattagt ttgagtttct aagagaatta ttgttgttag 720 gcttgtgtaa tttgtctaaa tatgttaaat ttctattaat taaagatagt aatctatttc 780 aatg 784 <210> 84 <211> 459 <212> DNA <213> Mytilus galloprovincialis <400> 84 agtgcgggta ataagagcct actcctcatt tgtgcataca tcttgaatgt tgttagggct 60 tacatgatca agggtagttt tcgtagggta ctttgcagtc tataggttat cggtagggct 120 gtttttctac gggtgctcgt taataaacaa gataagaata ggtagccagc taagtagggc 180 tgctagcggt atggggttgt taatactaat agggatgccc ccgtttcttg gtttcttagc 240 aaaagtattg gtatttttga tgaggggaag gcctgtaatt gtggcatgca tcataggatc 300 agtcattaga ttaaaatttt atattgattt cttttacaga atggtaataa aaagacctgt 360 ggacaaaaat aaggtagaag ttaaagccat ttggagtctg gtgatttgca taaacattat 420 gggggggggc gctgatttta gtaagattta tttagaatg 459 <210> 85 <211> 478 <212> DNA <213> Mytilus galloprovincialis <400> 85 agaggtggtg gtggtggtgg aatgagaggt cgtggacgag ggccaccaag aggaagacga 60 taaactcttt tatgtgtaaa atcaaagaca aagtgaaagt agaaaggaaa tctacagcta 120 tatattgttt tttgaccaac agcaaaaaat tatgttgtaa ctttctttta ttcttttgtt 180 ataatattcc tgataacatg cagacatgaa gaaacactat tcttttgtga aattttttca 240 agtattctca aaatcaggaa ataaacagat gaagataagt acgaaagaca tgaagcaatt 300 ccacatggac atattttaat catgtacatt cttctcattt ttaatgtcac atcatgtttc 360 atctccgtcg tcatttttgt acagaaataa aacaaaataa aaaaaattat ttcaaaaccc 420 caaacaaaaa caaaaaataa acccccatcg tagtgaattc attcacagaa tcactagt 478 <210> 86 <211> 197 <212> DNA <213> Mytilus galloprovincialis <400> 86 gaaaaacgat attgaaaaaa ccccaaccag acgggacaaa cgatgggaaa aacagtaaaa 60 gcggacacgg ccaaggctgg tgtcgaggga ggattaacat ttgatgaagt gtagagcgta 120 gtatgcccat tccggcgtgc atggctccga cttttacctc ccacggcacc tcggcttttc 180 ggtcgatacc gatacgg 197 <210> 87 <211> 555 <212> DNA <213> Mytilus galloprovincialis <400> 87 tacatcttca ggagctattg ctggagctgt tgctactaaa acagcggcaa gtgctttcac 60 aggtgctctt ataggaggct tgtctgccgg aagtgttggg tctgtaatgg ctggaagtat 120 tgcgttagca tctgggacac tgtcatcaag ccctttaggt ttgttgaccg ttggaaccaa 180 ttcaaatgga aatcagataa cacacgattg ttggaaacaa gttgtacacg aagcatcaca 240 ggagccgtcc aatgggataa ttctgaaaga gttgatctct catccgaatg ctgctgacgt 300 aacagtaata ccatgtggta atcttccata tgtcgtcatt gagaataggt ggaatgaaaa 360 atttgaactc gaatatgttg cactacttga atctaacaaa ttgtttttac acgttcagcc 420 actctgaaac tgaaaaaagc cactacaaca cttataataa tatgaccgat gtaaacttta 480 atattgtaca gtagtataac atgtggacat attgtaaggt attaatttaa gataaagtaa 540 acggtgtgaa taaca 555 <210> 88 <211> 180 <212> DNA <213> Mytilus galloprovincialis <400> 88 taaggtaacc aatatctttc aaacaggagg taaataggat gaagacatct tttcaaaatg 60 atgtacaaca gatctggtag aaattcttga aacattagtg acttattttt ttacttcttt 120 ttcaaatgat aaaaaggtct tgtattgata tagaaatcca ataaaattat cttcactttc 180                                                                          180 <210> 89 <211> 517 <212> DNA <213> Mytilus galloprovincialis <400> 89 ccgacgttca tataatccaa tgtaaacata aacagagata taaatagata gcgaacttgt 60 gcaatcggag ttttctaggt ccgtttcata ttttacgtta acagaacatg ttaattaaga 120 aatagttgat gacagctata gatttgtttt catttaacca tgggttggaa attgatattc 180 aaacttccaa gcggcagcgt tagtttagtt taaacacgtc catttatagt ggatagggaa 240 acaagttatt acaactgata ttaatccctt tcccctttgc aggtgcaagt gctgccttgt 300 agcgacatta gactgtcatt tttcgaaatc tacaagggtg tcttttacgc gcaaaatata 360 tggctctctc tcgctctctc ttaccatggg tcagccattt atcgtaccct tccgacggac 420 tatcatcgtt tcctcaagac catactcgta aatggtctca aggcaggggc gtagctaccc 480 ggaggcacga cagcacgtgc atccacgttg ttttcac 517 <210> 90 <211> 624 <212> DNA <213> Mytilus galloprovincialis <400> 90 gctccaggca ttcgcttcat gggggaaggg cacgcgcata gtaaccttga gatggacgta 60 cctgcattta caaaaacccg atccaggcga ccttccggtt caattggatc acgtgattat 120 gaagaaatgt ctacgtcatc aggggtttcg ttgaatgaca gttatagcta ttcttatttc 180 gatgtttcag acaattcaga tgagttggaa tctgcagcga ggcgtcggcg catctcaggg 240 gacgtacatg gcaggaatca agacgatatt cacttgaaag aaatcagcgc tgtaatggca 300 ttgacctctc tcgcgagatc tcgtagtaga tacccgatta tgtagtcacg tgattatcct 360 ccatatttaa tgcattccat atgcatctgt gcctatgtta caaactagga ggactatata 420 ttgttcttaa ttgtttatac gttactatgg ttacaggaac tgcagggaaa gtgtttaaaa 480 tgtttagatt ttatttttaa tgttatttgt caaacgcatt ttaagcatgc gcaatgaatg 540 ttgttttgaa gatatgcatg aatggttgaa gatagtgtgt gtctgggaat ggaataaatt 600 tttattgtta tagcaactaa attt 624 <210> 91 <211> 434 <212> DNA <213> Mytilus galloprovincialis <400> 91 ggaacatgat atcctacata agaagtatac ataataatgg acccgaggac cgactgtggg 60 actttaggtg caggaaaaat aaatacagct caaacagatg cagttggagt ggatgggtca 120 atgactggga tgagacattt agatttcaat gtcgggacaa cggatacata gctggaatat 180 acagtcgtca tgacaacgga ccagaggaca gaagatatga attctactgc tgccaaacaa 240 gaggaaaagt ctatgtccac agttgttatt atactaatcc taacgggttt gatagagaaa 300 tacgagagac agttccttat ggctatgcct ggaaaggagt tataagtaat catgataacg 360 gaccagagga ccgccagttt tccttccagc tttgccgaat gaactaataa cgatgaataa 420 agttatacaa taat 434 <210> 92 <211> 436 <212> DNA <213> Mytilus galloprovincialis <400> 92 gaccctacgc ctccggctac ggtcaccgtt gtaagcagtc cgggtacgaa cacgatgggt 60 ccagagacgc aggcaaaaat cagtcgcacg gtactagcgt tggtatcatc gcatctgaca 120 cgtctcgcaa aggtcgcccc ggtctcaacc gggaaacggc cggcgcacgg aagaaaggct 180 gtcgtcgacc aaactggggg tccccccggc acagtcggca caggtacaca cacgggaaaa 240 acgatattga aaaaaaaaac cccaaccaga cgggacaaac gatgggaaaa acagtaaaag 300 cggacacggc caaggctggt gtcgagggag gattaacatt tgatgaagtg tagagcgtag 360 tatgcccatt ccggcgtgca tggctccgac ttttacctcc cacggcacct cggcttttcg 420 gtcgataccg atacgg 436 <210> 93 <211> 221 <212> DNA <213> Mytilus galloprovincialis <400> 93 gggggggatt gagatgcctg aggagaaagc tgtgctttcc ggcatagttt gctgtttgga 60 aaggaaataa tatcattaat ttttggcccc aacaaaaatc acgaatcaaa aatttgaatt 120 cagataagca attaacaatc agacaaagat atttcaaatt atgttgactt aatttaattt 180 caaaatagtt aataaattga tgtagggata aaaaaaaaaa a 221 <210> 94 <211> 671 <212> DNA <213> Mytilus galloprovincialis <400> 94 gattgtgatg acgaatggca attggtgttc agagcacagc aaggaaatgg ccagggtgtt 60 tacaatgaat ggaagaggag atctccaagc acatgtgttg tctcctcagg ctgtttaccc 120 gaaaatgtcg atctggcaat gatgaaaata ccaaccaata aacatctcaa aagccctgta 180 atagacaaat ggtttttttc aaatatcaaa caggtgaaac tggaattatt tgacaacggt 240 aaatcagtga tgtatatgat ttttgatggc aagaacacaa actatttggg ttggtttttt 300 tacacaaaat taatagagag taattatgat gatctttaca gggatggttg tttttataag 360 ttccagttca gggactatta cagatattcc ttcatggtta aacaagacgg tcacgcttgt 420 gacacctcgg cacggggttg gctttcagtc attgattcac tttacagtag ttcctatagt 480 tacgcaagca cctgttttgg aatgagagtt agcagttatc caattttttt gtatgataag 540 aacaaccaca aaacaaggtg gtataataaa caatatggcc aaggtgatgt actcgccgta 600 tatgtcaaga gagtttaatc cagaccagat tcgaaccaat tgatataata aagattattg 660 cattttaaaa a 671 <210> 95 <211> 400 <212> DNA <213> Mytilus galloprovincialis <400> 95 gtagaggtgg tggtggtggt ggaatgagag gtcgtggacg agggccacca agaggaagac 60 gataaactct tttatgtgta aaatcaaaga caaagtgaaa gtagaaagga aatctacagc 120 tatatattgt tttttgacca acagcaaaaa attatgttgt aactttcttt tattcttttg 180 ttataatatt cctgataaca tgcagacatg aagaaacact attcttttgt gaaatttttt 240 caagtattct caaaatcagg aaataaacag atgaagataa gtacgaaaga catgaagcaa 300 ttccacatgg acatattata atcatataca ttcttctcat ttttaatgtc acatcatgtt 360 tcatctccgt cgtcattttt gtacagaaat aaaacaaaat 400 <210> 96 <211> 285 <212> DNA <213> Mytilus galloprovincialis <400> 96 attgaaatgg atttattgat ttaaatttaa atttcgaatc cattgaatga agtccattcc 60 ccggcatagc agtttcctcc accagttcgt tccattctta tccaaatttt gtcatgtttc 120 ttcaaagaaa ggacacccga ctgtgttgat gaatggttcc gcttactacc ttcattccaa 180 gtatgatttg ctaacccaac aggattacca ttgtgcacaa gttcagtagg acagcttcca 240 ccagcgtttg tatacattgt ccaggaaaag taaaatgtac catca 285 <210> 97 <211> 343 <212> DNA <213> Mytilus galloprovincialis <400> 97 gcagataaca gcagtagact tcaatcatgc ttgaatgtct gcgcaaacat gaaacaatgg 60 aagttaggta gtgataatga gtctcagtct gatgcagtcg ctaggcaaca aaaagcctgt 120 gagagtcgtt gcaacaaccg ctttggggca ggatcatcat aatactatgt ggaatgacag 180 gatgtcaact actgttggcc aaaattcaac tcttcgtgac aatcatcata cggcaacaca 240 aattagtctt tgaattctga aattggatat ggattattca tttgatttga tttcactcat 300 gtccaactgc ataaaattca gcaataaatt gttttgaaat atg 343 <210> 98 <211> 662 <212> DNA <213> Mytilus galloprovincialis <400> 98 agatgaccca ggcaaatttt ttgttggaaa cctgagctgg gatacagaca atgacagttt 60 atggaactac ttcagtcagt atggacagct gtcagatgct gttgtaatca tggacagaca 120 aactggaagg tccagaggtt ttggttttgt gacattcaga gatccaagat gtgtggactc 180 agtattacat caacaacaac acgaaattga tggaagacag atcagtgcca gaccttgcaa 240 cagccgatag atagactgtt tatggagttg aagtggaaaa aacaaattta aaatcatgtg 300 tattgacacc taacttgtgg tgtatataaa tctccatgat ttgtaactat gtattgtttg 360 ttgtattgaa aattccattt gtttttaaat ataaaaactt ctcattttat acaactgtcc 420 ttcatttgtt tttaaatata caaacttctc attttatacc attgcattgt gtactttgta 480 acagctaaaa aattttatta acaaatgtaa tgataataat tagtgtttta acattggagc 540 aaagttatta gtgtatccaa taaaagaata aattaaaaat gttagaataa caacattaat 600 ttcatgtatt ttaattgatc actgtcttca gatgaagtaa attatataaa gtctttgaaa 660 ct 662 <210> 99 <211> 403 <212> DNA <213> Mytilus galloprovincialis <400> 99 atgtagaaga ccggaatgga aaaaagcatt atgcagttta tagaaaattc tcaataggcg 60 acgcatctac aaaatacaga ttaagtatca gcgattactc aggaaatgca ggggacggat 120 tgagctattt caatgggatg aagttctcaa catatgatca agataacgac agagtaagta 180 atatgaactg tgctgataat ccccacttaa aaggtggatg gtggtacaat aactgctgga 240 gaagtactga cgcaatgcta aatggacatt acaccggtaa aaatagttct aaacagggta 300 taagatatcg ttcgcttgga caacatactc agaaaaaatc aactatgaag attcgaaaag 360 tgtaattaca tgtctagcta ccataaataa atggaagcaa aat 403 <210> 100 <211> 459 <212> DNA <213> Mytilus galloprovincialis <400> 100 agtgcgggta ataagagcct actcctcatt tgtgcataca tcttgaatgt tgttagggct 60 tacatgatca agggtagttt tcgtagggta ctttgcagtc tataggttat cggtagggct 120 gtttttctac gggtgctcgt taataaacaa gataagaata ggtagccagc taagtagggc 180 tgctagcggt atggggttgt taatactaat agggatgccc ccgtttcttg gtttcttagc 240 aaaagtattg gtatttttga tgaggggaag gcctgtaatt gtggcatgca tcataggatc 300 agtcattaga ttaaaatttt atattgattt cttttacaga atggtaataa aaagacctgt 360 ggacaaaaat aaggtagaag ttaaagccat ttggagtctg gtgatttgca taaacattat 420 gggggggggc gctgatttta gtaagattta tttagaatg 459 <210> 101 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 101 ggtcggtgaa acatcaatc 19 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 102 agaatacaag taccgaaagg 20 <210> 103 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 103 ggtactagcg ttggtatcat c 21 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 104 gtccgctttt actgtttttc 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 105 gtgggatgat gttgatatag 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 106 ccattgccta tagcatttac 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 107 cagggaagtt gtaatatctg 20 <210> 108 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 108 tactctgaaa acacaggtga c 21 <210> 109 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 109 gtgtcttctt atgggtctg 19 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 110 gctataaaca tgctttctcc 20 <210> 111 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 111 tggacagagt ttaaaggag 19 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 112 ctagcataga cttttggttg 20 <210> 113 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 113 acagtcggca caggtacac 19 <210> 114 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 114 gcatactacg ctctacactt c 21 <210> 115 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 115 caggttttac tgctacagg 19 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 116 gttaatcacg atcccttgtg 20 <210> 117 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 117 ggtgactgtt tttatggtc 19 <210> 118 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 118 cgaagagaat gaaatgtcc 19 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 119 ggttgctgat aaagtagttg 20 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 120 attcagtctg gtcttctttc 20 <210> 121 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 121 atatgtagaa gggagttgg 19 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer sequence for real-time quantitative PCR <400> 122 atctaactcc tatggtcctc 20

Claims (18)

A microarray chip for stress detection or medical examination of marine ecosystems, wherein the nucleic acid sequence oligonucleotides or complementary strand molecules of any one or more genes selected from the following groups are integrated:
60S ribosomal protein L32 protein mRNA, 40S ribosomal protein S3, 60S ribosomal protein L3 and related proteins, Ribosomal protein L11 mRNA, Elongation factor 2 mRNA, 40S ribosomal protein S2 putative mRNA / 30S ribosomal protein S5, 60S acidic ribosomal protein P2 (Genbank Accession FM165434.1), 40S ribosomal protein S4, Nucleolar complex associated 2 homolog, 40S ribosomal protein S28, 60S ribosomal protein L21, Elongation factor TU (tufA) gene, EF1-alpha mRNA for elongation factor 1 alpha (Genbank Accession No. EU684228.1), RecA gene, Zinc finger NFX1-type containing 1, tRNA-Leu (trnL) gene, Molecular chaperone (HSP90 family), Prefoldin subunit 1 putative mRNA, DnaJ domain containing protein mRNA, Hsp70-1 gene (Genbank Accession No. AJ585375.1), Aspartate beta-hydroxylase (ASPH), Matrix metallopeptidase 9, Heat shock protein 12B, C-type lectin mRNA, LIM protein (LIM) mRNA, Low-density lipoprotein receptors containing Ca2 + -binding EGF-like domains , Surfactant protein A, Calcium-dep endent protein kinase, Calcium / calmodulin-dependent protein kinase II alpha, GTPase-activating protein GYP 7, Ral-GDS related protein Rgr, CEGP1 protein, MEGF10 protein, 18S ribosomal RNA gene (Genbank Accession No. DQ640546.1), 16S ribosomal RNA gene (Genbank Accession No. AF023552.1), Fibrillar collagen, Phosphate regulatory protein genes phoB-phoR, Galactose-binding lectin, Lec4 lectin mRNA, Tubulin beta 2A, microtubule associated protein (RHAMM) mRNA , Chitinase mRNA, Sodium-dependent phosphate / anion cotransport, Endo-1,3-beta-glucanase Engl1, Organic anion transporter, Phosphoglycerate / bisphosphoglycerate mutase family protein, Peptidoglycan glycosyltransferase, ABC transporter AbcG16, Extracellular Cu / Zn-superoxide dismutase mRNA ( Genbank Accession No. FM177867.1), Immunoglobulin kappa chain complex (Igk), 6-deoxyerythronolyde B synthase II, Polyketide synthase gene cluster, Farnesyl diphosphate synthase, Gamma-glutamyltransferase 8 pseudogene, Cytochrome P-450 hydroxylase homolog (nidi) gene, Anthranilate N-methyltransferase mRNA, Mitochondrial ubiquinol-cytochrome c reductase iron-sulfur subunit, Cytochrome oxidase subunit I gene (Genbank Accession No. GQ480294.1), NADH: ubiquinone oxidored uctase NDUFA2 / B8 subunit, ATP synthase F0 subunit 6, Sn-glycerol-3-phosphate dehydrogenase, NADH dehydrogenase subunit 4 (Genbank Accession No. AF315186.1), Flavin containing monooxygenase 3 (FMO3) gene, Guanine nucleotide-binding protein subunit beta-like protein 1 (gnb1l) mRNA, Carcinolectin5b-5, Rhamnose-binding lectin, Ubiquitin-protein ligase E, Sialic acid binding lectin, Fertility restorer-like protein, Somatic embryogenesis receptor-like kinase 2, Transcriptional activator, TenA family, Transcription antiterminator, BglG family, Transcriptional regulator, LacI family, Leucine-rich repeat kinase 2, Transcriptional regulator, TetR family, SH2 domain containing 4B ( SH2D4B), Ecotype Hr-5 disease resistance protein, Collagen protein, MHC class II antigen beta (Genbank Accession No. AJ249992.1), Interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), Mytilin C precursor, Endoproteinase Arg-C precursor , Apextrin, Neuromacin, Matrix protein (gag), Opticin (Optc) gene, Glycopeptide AFGP polyprotein precursor gene, Cadherin 11 (Cdh11) gene, Microsatellite Myco-14 sequence, Proteasome beta 9 subunit gene, Hemagglutinin / ameboc yte aggregation factor precursor putative mRNA, Alpha 10 subunit of nicotinic acetylcholine receptor, MFN2 gene for mitofusin 2, Rendezvin (RDZ) mRNA, Calnexin (pp90) mRNA, Interleukin-20 receptor alpha, Interferon regulatory factor 7 (IRF7) mRNA, DAZ- associated protein 1 putative mRNA, Cytotactin and NADH -ubiquitin oxidoreductase chain 2.
The microarray chip according to claim 1, wherein the nucleic acid sequence oligonucleotide of any one or more genes selected from the following group or a complementary strand molecule thereof is integrated:
18S ribosomal RNA (Genbank Accession No. DQ640546.1), ALPHA 10 nAChR gene for alpha 10 subunit of nicotinic acetylcholine, EF1-alpha mRNA for elongation factor 1 alpha (Genbank Accession No. EU684228.1), chitinase mRNA, cytochrome oxidase subunit 1 (cox1) (Genbank Accession No. GQ480294.1), NADH dehydrogenase 1 alpha subcomplex subunit 2 putative mRNA, calcium / calmodulin-dependent protein kinase II alpha, GTPase-activating protein GYP 7, phosphoglycerate / bisphosphoglycerate mutase family protein, hsp90, ATP synthase F0 subunit 6.
The method according to claim 1, wherein the gene is Mediterranean sea mussel ( Mytilus galloprovincialis ) microarray chip.
The microarray chip according to claim 1, wherein the gene is increased or decreased in expression by the amount of dissolved oxygen.
The microarray chip according to claim 1, wherein the stress is a change of dissolved oxygen in seawater.
5. The microarray chip according to claim 4, wherein the amount of dissolved oxygen is caused by the formation of hypoxia lumps.
The microarray chip of claim 1, wherein the microarray chip detects a marine ecosystem response to a change in dissolved oxygen.
1) separating RNA from mediterranean mussels suspected of being exposed to the stress of the marine ecosystem of the experimental group and normal mediterranean mussels of the control group;
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 or 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 8, 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 (rhodamine). Stress detection or medical examination method of marine ecosystem.
9. The method of claim 8, wherein the stress is a change in the amount of dissolved oxygen in the seawater.
The method of claim 10, wherein the change in the amount of dissolved oxygen is due to the formation of hypoxia (hypoxia) water mass.
9. The method of claim 8, wherein the method detects marine ecosystem responses to changes in dissolved oxygen content.
1) separating RNA from mediterranean mussels suspected of being exposed to the stress of the marine ecosystem of the experimental group and normal mediterranean mussels of the control group;
2) Quantitative real-time reverse transcript polymerase chain reaction (qRT) using RNA pairs of step 1) using primer pairs complementary to the genes integrated in the microarray chip of claim 1 and amplifying the genes. -PCR); And
3) Stress detection or 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 stress detection or health diagnostic kit of the marine ecosystem comprising the microarray chip.
15. The kit of claim 14, wherein said stress is a change in dissolved oxygen amount.
16. The kit of claim 15, wherein the amount of dissolved oxygen is due to the formation of hypoxia (hypoxia) water masses.
15. The kit of claim 14, wherein the kit detects marine ecosystem responses to changes in dissolved oxygen content.
Claim 1 kit for stress detection or health diagnostics of marine ecosystems comprising a primer pair complementary to the gene integrated in the microarray chip of claim 1 and capable of amplifying the gene.

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