KR20170025324A

KR20170025324A - Ocean Acidification responsive genes in Dendronephthya gigantea and the method for diagnosing marine ecosystem using the same

Info

Publication number: KR20170025324A
Application number: KR1020150121637A
Authority: KR
Inventors: 송준임; 우선옥
Original assignee: 이화여자대학교 산학협력단
Priority date: 2015-08-28
Filing date: 2015-08-28
Publication date: 2017-03-08

Abstract

The present invention relates to a gene derived from Dendronephthya gigantea corresponding to a change in acidity of seawater and a method for diagnosing a marine ecosystem using the same. Particularly, Therefore, they can be usefully used as microarrays and kits which can confirm exposure to acidified sea water by changing the acidity of seawater by using them as biomarkers.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for diagnosing marine echinocarcinogenesis,

The present invention relates to a process for the preparation of a large resin marble ( Dendronephthya gigantea gene and a method for diagnosing acidification of a marine ecosystem using the same.

Since the Industrial Revolution, humans have increased atmospheric carbon dioxide concentrations by more than 100 ppm, and the oceans are thus a very important sink for carbon dioxide, absorbing about a quarter of the additional carbon dioxide released into the atmosphere. Carbon dioxide absorbed into the ocean will form carbonate (H ₂ CO ₃ ) in seawater, and additional absorbed carbon dioxide increases the acidity of seawater.

As the acidity (hydrogen ion concentration) of seawater increases due to oceanic acidification, the concentration of carbonate ion (CO ₃ ^2- ) decreases, so it has a great influence on marine organisms composed of carbonate skeleton. In the future, when the oceanic acidification causes the carbonate ion concentration in the surface sea water to steadily decrease to the carbonate unsaturation state, the species using the carbonate skeleton are likely to become extinct without sustaining any further life activity

The effects of acidification of marine organisms in the pre-oceans have been extensively perceived, and in this trend it is predicted that within a few centuries the acidification will destroy the coral reefs in the tropical oceans and skeletal formation of calcified organisms will be difficult in most polar waters.

The amount of carbon dioxide emitted by mankind during the year was 31.6 billion tons, an increase of about 1 billion tons compared to 2010, and if the amount of carbon dioxide exceeds the ecosystem critical point, the ecosystem will change seriously. Recently, rapid acidification has been reported in the East Sea of Korea after the Industrial Revolution. This suggests that marine acidification is also proceeding seriously in the offshore waters of Korea, suggesting that the habitat of biomass with calcium carbonate skeleton may be reduced in the future. Therefore, it is urgent to develop a biological forecasting system that can detect ecosystem change at a local scale in advance.

Although corals are used as habitats for various marine life, species diversity and population are rapidly decreasing due to rising sea water temperature and acidification due to climate change. Habitat destruction due to human interference is rapidly progressing In addition to ecological monitoring for conservation of coral reefs, a research approach at the molecular level is needed.

Big resin bream (Scientific name: Dendronephthya gigantea ) is a kind of gypsophila which is taxonomically distributed in the zoophyllous animal, coral reef river, It is found mostly in the rocky basin around 5m depth and inhabits a somewhat strong current. There are many variations in color of the colony, such as purple, red and pink, and they grow to a maximum height of over 30cm, which plays a major role in the coastal marine biodiversity. This coral species has been designated as a statutory animal by the Ministry of Environment's list of notifications (Goshi-jin) and is a biological resource subject to approval for export from abroad.

Studies on coral reefs in Korea have been proceeding from the taxonomic studies of 1970s to the systematic, evolutionary, symbiotic, life history, proliferation and conservation. However, studies on ocean acidification and climate change are still in the beginning stage.

The ecosystem health assessment method using the gene expression changes can be applied to various environmental factors in the environment by applying the molecular technology that is developing at a very high speed to the molecular biology technology It is a method to evaluate the risk or the health.

Existing methods for assessing the presence of harmful organisms (such as mutation, behavioral abnormality, mortality, and enzyme activity) using biological organisms are methods of measuring the outcome of organisms that have already been exposed to stressful substances, It has a weakness that it can be measured at a fairly high level. On the other hand, the evaluation method using the gene expression change is a method of evaluating the level of stress the organism is receiving by using the change in the transcription level that the gene information is passed to the protein after receiving the external stimulus, It is also possible to predict the outcome of biological reactions when exposed to a small amount of environmental change or long-term exposure to external stimuli. The gene biomarkers used in the evaluation method using these gene expression changes can tell the existence and the relation of the external stimulus through the specificity thereof, and the long-term effect on various environmental changes of the ecosystem and the health However, there is no known method for determining the degree of oceanic acidification using large resin buds.

The inventors of the present invention have identified a change in the expression level of a specific gene by ocean acidification in a large resin beetle, which plays a large role in the maintenance of marine biodiversity while extracting biomarkers capable of detecting ocean acidification, The present inventors have completed the present invention by confirming that the health diagnosis and the degree of acidification of the sea water can be grasped by using the gene.

An object of the present invention is to provide a gene derived from Dendronephthya gigantea corresponding to marine acidification, a method for confirming acidification of marine ecosystem using the same, or a method for diagnosing environmental change of marine ecosystem.

In order to achieve the above object, the present invention provides a microarray for confirming exposure to acidified sea water in which oligonucleotides or complementary strand molecules of a nucleic acid sequence of any one or more genes selected from the following groups are integrated do:

(L-Asparaginase), a gene described in SEQ ID NO: 3 (Cadherin EGF LAG seven-pass G-type receptor), SEQ ID NO: 4 (DNA-directed RNA polymerase III subunit), the gene described in SEQ ID NO: 6 (Dynactin subunit 1), the gene described in SEQ ID NO: 7 (FAD -dependent oxidoreductase), Ficolin (SEQ ID NO: 8), Myosin light polypeptide 6, Phosphoenolpyruvate carboxykinase (SEQ ID NO: 10), Gene A polyketide synthase, a gene described in SEQ ID NO: 13 (Protein phosphatase 1 regulatory subunit 12A), a gene described in SEQ ID NO: 14 (Pyrroline-5-carboxylate re the gene described in SEQ ID NO: 16 (Alcohol dehydrogenase 1), the gene described in SEQ ID NO: 17 (Aquaporin TIP2-3), the gene described in SEQ ID NO: 18 A gene described in SEQ ID NO: 19 (Guanidinoacetate N-methyltransferase) or a gene described in SEQ ID NO: 20 (Protein tyrosine phosphatase type IV A3).

The present invention also provides a method for determining whether an acidified sea water is exposed, comprising the steps of:

1) isolating the RNA from the experimental group exposed to acidified sea water, Dendronephthya gigantea , and the control group, large resin bream ;

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

3) hybridizing the cDNAs labeled with different fluorescent substances of step 2) with the microarray of the present invention;

4) analyzing the reacted microarray; And

5) From the analyzed data, the degree of gene expression integrated in the microarray of the present invention is compared with the control group.

Also, the present invention provides a kit for confirming exposure to acidified sea water including the microarray according to the present invention.

In addition, the present invention provides a kit for confirming exposure to acidified sea water containing a pair of primers complementary to each gene and capable of gene amplification for the genes according to the present invention.

The present invention relates to a gene derived from Dendronephthya gigantea corresponding to a change in acidity of seawater and a method for diagnosing a marine ecosystem using the same. Particularly, Therefore, they can be usefully used as microarrays and kits which can confirm exposure to acidified sea water by changing the acidity of seawater by using them as biomarkers.

Figure 1 is a grouping of common genes among genes that are differentially expressed more than 2 times as a result of microarray experiment with a control group (pH 8.0) and experimental groups (pH 6.5, 7.0 and 7.5).

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have found that a large resin mambrum ( Dendronephthya gigantea ) was excavated. Therefore, a microarray in which the expression level of the large resin-bound mammary gland-derived gene is accumulated in response to changes in the acidity of seawater can be used for detecting acidified sea water exposure and for diagnosing the health status of a marine ecosystem.

The present invention provides a microarray for confirming whether or not an oligonucleotide or a complementary strand molecule of a nucleic acid sequence of any one or more genes selected from the following group is accumulated on acidified sea water:

It is preferable that the gene is derived from the Kokugo, and it is more preferable that the gene is derived from a large resin beetle.

The pH of the acidified sea water is preferably 6 to 7.7, more preferably 6.5 to 7.5, but is not limited thereto.

1) isolating the RNA from the large resin beetle, which is an experimental group exposed to acidified sea water, and the large resin beetle, which is a control group;

4) analyzing the reacted microarray; And

The fluorescent material of step 2) may be selected from the group consisting of Cy3, Cy5, polyL-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate isothiocyanate, RITC) and rhodamine, but the present invention is not limited thereto.

2) Performing Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a primer pair complementary to the following genes and capable of gene amplification, using the RNA of step 1) as a template,

(L-Asparaginase), a gene described in SEQ ID NO: 3 (Cadherin EGF LAG seven-pass G-type receptor), SEQ ID NO: 4 (DNA-directed RNA polymerase III subunit), the gene described in SEQ ID NO: 6 (Dynactin subunit 1), the gene described in SEQ ID NO: 7 (FAD -dependent oxidoreductase), Ficolin (SEQ ID NO: 8), Myosin light polypeptide 6, Phosphoenolpyruvate carboxykinase (SEQ ID NO: 10), Gene A polyketide synthase, a gene described in SEQ ID NO: 13 (Protein phosphatase 1 regulatory subunit 12A), a gene described in SEQ ID NO: 14 (Pyrroline-5-carboxylate re the gene described in SEQ ID NO: 16 (Alcohol dehydrogenase 1), the gene described in SEQ ID NO: 17 (Aquaporin TIP2-3), the gene described in SEQ ID NO: 18 A gene described in SEQ ID NO: 19 (Guanidinoacetate N-methyltransferase) or a gene described in SEQ ID NO: 20 (Protein tyrosine phosphatase type IV A3); And

3) comparing the gene product of step 2) with the control group to confirm the expression level.

The gene described in SEQ ID NO: 1 to SEQ ID NO: 15 has increased expression as compared with the control group, and the gene represented by SEQ ID NO: 16 to SEQ ID NO: 20 has decreased expression as compared with the control.

The kit may further include any one selected from the group consisting of streptavidin-like phosphatase conjugate, chemifluorescent, and chemiluminescent fluorescent substance, But is not limited thereto.

In addition, the kit may be any one selected from the group consisting of a buffer solution used for hybridization, a reverse transcriptase for synthesizing cDNA from RNA, a reaction reagent group consisting of dNTP and rNTP (premixed or separated feed type), a labeled reagent, and a washing buffer solution But it is not limited thereto.

In addition, the present invention provides a kit for confirming exposure to acidified sea water comprising a pair of primers complementary to each of the following genes and capable of amplifying the gene:

It is preferable that the primer is at least one selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 60.

In a specific embodiment of the present invention, in order to discover a large resin-bound mammary gland-derived gene corresponding to a change in the acidity of seawater, a large resin beetle is cultured in seawater at various pH, and then the cultured large resin-bound mammalian cDNA is synthesized The genes whose expression levels were changed were examined. Specifically, mRNAs were isolated from the tissues of large resin buds cultured at pH 6.5, pH 7.0 and pH 7.5, respectively, and cDNA was synthesized using this as a template. The synthesized cDNA was fluorescently labeled with Cy3-CTP and Cy5-CTP, and a microarray was constructed using the fluorescently labeled Cy3-CTP and Cy5-CTP. The prepared microarray was scanned using a Axon GenePix 4000B scanner (Axon Instrument, USA) and analyzed. As a result, 15 genes (see Table 1) and 5 genes (see Table 2) whose expression levels were increased in the experimental group were confirmed based on the control group. In addition, the common genes among the above genes differentially expressed in the experimental group 2 times or more were grouped (see FIG. 1). Climate change causes changes in the acidity of seawater, and with changes in these environments, marine organisms cause changes in physiology and metabolism. Therefore, it can be used as a biomarker for confirming the stress and health condition due to changes in the external environment by confirming the change in the expression amount of the gene derived from the marine organism.

Therefore, the genes can be usefully used as microarrays and kits for confirming exposure to acidified sea water according to changes in acidity of seawater.

Hereinafter, the present invention will be described in detail with reference to examples.

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

< Example 1> Big Resin ( Dendronephthya gigantea ) And exposure to acidified sea water

<1-1> Big resin culture

The present inventors sampled large resin drums in Jeju coastal waters and cultured them in a laboratory.

Specifically, a large resin bream collected from the coast of Jeju was cultured in natural seawater through three kinds of filters of 100, 10 and 1 ㎛. At this time, the water temperature was fixed at 24 ° C using an underwater heater, and the water temperature was adjusted to 14:10.

<1-2> Exposure to acidified sea water

The large resin buds cultured by the method of Example <1-1> were exposed to acidified sea water.

Specifically, large resin buds were divided into three groups and cultured in seawater at pH 7.5, pH 7.0 and pH 6.5 for 48 hours, and the control group was cultured in natural sea water (pH 8.0) for 48 hours. In addition, candidates of the pink sea mantram gene of the Korean sea bream were selected through the microarray experiment after acidification and exposure to sea water, and the candidate genes corresponding to the acidified exposure were selected by comparing the large resin mambrum gene sequences.

< Example 2> by acidification Big resin Gene change measurement

<2-1> Isolation of RNA from a large resin beetle

RNA was isolated according to the method developed by the present inventors in order to measure a change in the gene of the large resin beetle exposed to the acidified sea water by the method of the above Example <1-2>.

Specifically, the large resin mambr tami tissues of the experimental group and the control group were made into powder by using liquid nitrogen in a mortar and dissolved in a lysis solution (35 mM EDTA, 0.7 M LiCl, 7% SDS, 200 mM Tris-Cl 9.0)] was added and homogenized. The same amount of phenol solution was added to the homogenized sample, mixed well, and centrifuged for 10 minutes. The supernatant was transferred to a new tube, and 1/3 of the total volume of lithium chloride (LiCl) was added to the homogenized sample. And then left at 4 캜 for 2 hours or more. The supernatant was removed by centrifugation for 30 minutes, and the precipitate was collected and dissolved in 300 μl of DEPC-treated water. The same amount of isopropanol was added to 1/10 volume of 3 M sodium acetate (pH 5.2) After centrifugation for about 30 minutes, the supernatant was removed and the precipitate was taken. 50 μl of a 70% ethanol solution was added to the precipitate, followed by centrifugation for 5 minutes. Then, the ethanol solution was removed, the precipitated RNA was dried, and the dried RNA was dissolved in an appropriate amount of DEPC-treated water.

<2-2> Synthesis of cDNA

CDNA was synthesized from a large resin mammal RNA isolated by the method of Example <2-1> above.

Specifically, Tri-reagent (Molecular Research Center Inc., USA) was used for the isolation of specific genes corresponding to acidified seawater. Using the extracted whole mRNA (3.0 ㎍) as a template, reverse transcriptase was used to synthesize cDNAs of the control and experimental groups.

<2-3> cDNA sequencing

The cDNA synthesized in the above Example <2-2> was subjected to sequence sequencing directly using the next-generation sequencing technique, and the obtained nucleotide sequence was aligned to obtain a full map of the expression construct using the control genome .

Specifically, poly (A) RNA was obtained and the first strand was synthesized using oligo (dT) or random hexamer. Then, a second strand complementary to the first strand was obtained and cDNA synthesis was completed. The DNA double strand was partially cleaved, the deoxyadenine base was added to the 3 'end, the DNA was ligated using the illumina adapter, amplified by PCR method, and the amplified product was selected by size and sequenced.

<2-4> Hybridization (Hybridization) and scanning (scanning)

The present inventors prepared a large resin mandrel cDNA microarray and hybridized and scanned it.

Specifically, 1-2 μg of the cDNA was prepared in the experimental group and the control group, respectively. The cDNA was dissolved in 0.1 M sodium bicarbonate buffer (pH 8.7), mixed with 40 nm Cy3 and Cy5 fluorescent materials, . After 90 minutes, 15 [mu] l of 4 M hydroxylamine was added and left in the dark for 15 minutes. A large resin-bound cDNA sample labeled with a fluorescent substance was purified using a PCR purification kit (Qiagen, Germany) and eluted with distilled water. The purified fluorescent label-cDNA sample was added to a hybridization buffer (3x SSC, 0.3% SDS, 50% formamide, 20 Cot Cot-1 DNA, 20 ye yeast tRNA) 30 < / RTI > to make a hybridization mixture. The hybridization mixture was denatured by heating at 95 DEG C for 3 minutes and centrifuged at 12,000 g for 30 seconds to lower the temperature of the heated hybridization compound. The prepared large resin admiral cDNA microarray was covered with a coverslip and the modified hybridization mixture was pipetted. The microarray was placed in a GT-Hyb chamber and reacted at 65 ° C for 16 hours. After hybridization, the microarray was removed from the chamber and cleaned. The microarray was rotated, dried and stored in a dark room until scanning. The large resin micro-array of microarrays that had been tested was scanned using an Axon GenePix 4000B scanner (Axon Instrument, USA). In the GenePix Pro 6.0 program, each point from the scanned image is graded using a gridding file and quantified to include analysis values such as Cy5 / Cy3 intensity and ratio of each point. I got a GPR file (GPR file).

<2-4> Microarray Data analysis

From the GPR files obtained from the JeanPixPro 6.0 program, the following analysis was performed using the analysis program Jean Spring 7.3.1 (GeneSpring 7.3.1, Agilent Technologies, USA). Normalization was performed using LOWESS (Locally Weighted Regression Scatter Plot Smoothing), and the reliable gene showed that the sum of the median values was lower than the background, or the standard deviation of each pixel value was significant And flag-out the point where it is not. Significant genes were also selected to show a difference of more than two times the normalized ratio value.

As a result, as shown in Table 1 and Table 2, 15 genes (Table 1) significantly increased the expression level by more than 2 times in the acidification test group (pH 6.5, 7.0, 7.5) 2), and a total of 20 genes are shown in SEQ ID NOS: 1 to 20. In addition, as shown in Fig. 1, common genes among the above genes differentially expressed in the test group 2 times or more were grouped (Fig. 1).

Up-regulated gene lists and changes in acidified sea water number gene Change (x) One Acetyl-CoA hydrolase 6.82 2 L-Asparaginase 5.91 3 Cadherin EGF LAG seven-pass G-type receptor 15.34 4 Calpain small subunit 1 20.26 5 DNA-directed RNA polymerase III subunit 18.50 6 Dynactin subunit 1 28.53 7 FAD-dependent oxidoreductase 6.96 8 Ficolin 6.21 9 Myosin light polypeptide 6 5.40 10 Phosphoenolpyruvate carboxykinase 6.97 11 Phosphoserine aminotransferase 6.22 12 폴리켓이드 synthase 11.56 13 Protein phosphatase 1 regulatory subunit 12A 4.59 14 Pyrroline-5-carboxylate reductase 6.57 15 transferrin receptor protein 2 7.29

List and change of down-regulated genes in acidified sea water number gene Change (x) One Alcohol dehydrogenase I -2.44 2 Aquaporin TIP2-3 -2.26 3 Deoxyribose-phosphate aldolase -2.30 4 Guanidinoacetate N-methyltransferase mRNA -9.30 5 Protein tyrosine phosphatase type IV A3 -11.84

The primer for detection of the gene whose expression was regulated in the acidified sea water of the present invention and the PCR product size number gene primer PCR product size (bp) One Acetyl-CoA hydrolase F-AGATGAAGCATAACCCTCAA (SEQ ID NO: 21)
R-CTTAAGCTGGCAGACTTTTT (SEQ ID NO: 22) 188 2 Alcohol dehydrogenase I F-AAACAAGCCACTCTGAAAGA (SEQ ID NO: 23)
R-CTTTCGGAAATCTTCATCAG (SEQ ID NO: 24) 249 3 Aquaporin TIP2-3 F-AGTCACAGCTTGAAACCACT (SEQ ID NO: 25)
R-CGTATTCTGGTACGCATCTT (SEQ ID NO: 26) 251 4 Cadherin EGF LAG seven-pass G-type receptor F-CAGAGGAATGTTTCTTCGAG (SEQ ID NO: 27)
R-TTGTTGGAGAAGCAGTAGGT (SEQ ID NO: 28) 205 5 Calpain small subunit 1 F-ACGACAAGGACTACAGCATT (SEQ ID NO: 29)
R-CCATAGACCAGCCAGTTTAG (SEQ ID NO: 30) 230 6 Deoxyribose-phosphate aldolase F-AACAAGAGTGCGTTGAAGTT (SEQ ID NO: 31)
R-TGAGATTTTTGTAGGGATGC (SEQ ID NO: 32) 178 7 DNA-directed RNA polymerase III subunit F-CGGACTACTTCAAGACGAAC (SEQ ID NO: 33)
R-CCGTGGTTGAGTGAGTAAGT (SEQ ID NO: 34) 238 8 Dynactin subunit 1 F-AAGAAAGGAAAGCCAGAACT (SEQ ID NO: 35)
R-TGGTGGAGGAGTTAGAAGAA (SEQ ID NO: 36) 220 9 FAD-dependent oxidoreductase F-CGTTTCTTACAGCTCGTTCT (SEQ ID NO: 37)
R-ACTTTTCCACAACAGCAACT (SEQ ID NO: 38) 230 10 Ficolin F-CTACTCCACTTTCGAGTCGT (SEQ ID NO: 39)
R-CTCTTGTGGAACTCCAGATT (SEQ ID NO: 40) 174 11 Guanidinoacetate N-methyltransferase F-ACAGTTCCCTCAATGATTTC (SEQ ID NO: 41)
R-GTTATTGGGTTGATCAGGAA (SEQ ID NO: 42) 200 12 L-Asparaginase F-ATCACTGACCCTCAATGTTC (SEQ ID NO: 43)
R-TTCCCTCAGAGTGTCTGTTC (SEQ ID NO: 44) 295 13 Myosin light polypeptide 6 F-CTGAGTTCCAAGACTCCAAG (SEQ ID NO: 45)
R-AGATCACCTTGCACTGATTC (SEQ ID NO: 46) 174 14 Phosphoenolpyruvate carboxykinase F-ACACCAGGTACAAGATGCTC (SEQ ID NO: 47)
R-CTCCTTCTCCTTGATCTCCT (SEQ ID NO: 48) 203 15 Phosphoserine aminotransferase F-AATATCTTGCATTGGGATTG (SEQ ID NO: 49)
R-CGCAAATTTCTTTCTGTCTT (SEQ ID NO: 50) 199 16 폴리켓이드 synthase F-ACACAGCAGAAACAAGAGGT (SEQ ID NO: 51)
R-CATCTTCATTCAACCCAAAT (SEQ ID NO: 52) 213 17 Protein phosphatase 1 regulatory subunit 12A F-AATACCAGTCAGTGGTCAGG (SEQ ID NO: 53)
R-AAGTCTCCAAATGTCTCCAA (SEQ ID NO: 54) 229 18 Protein tyrosine phosphatase type IV A3 F-ATCAAAGCGCAGGAGAAG (SEQ ID NO: 55)
R-GAAGTCCACCCTTTCAGTG (SEQ ID NO: 56) 206 19 Pyrroline-5-carboxylate reductase F-TTTAGCCACTTCAGGACAAT (SEQ ID NO: 57)
R-GTTGTCTGAAATCGGAGTTG (SEQ ID NO: 58) 191 20 transferrin receptor protein 2 F-CCTCGAACACTGAAGGACTA (SEQ ID NO: 59)
R-GGCACTGCTACTGAAGATGT (SEQ ID NO: 60) 214

<110> Office of Reseach / Ewha University-Industry Collaboration Foundation <120> Ocean Acidification responsive genes in Dendronephthya gigantea and the method for diagnosing marine ecosystem using the same <130> 2015P-06-002 <160> 60 <170> Kopatentin 2.0 <210> 1 <211> 273 <212> DNA <213> Dendronephthya gigantea <400> 1 ccaatcagat cgctccggcg gcagatgaag cataaccctc aatgcatcgg catgtccgca 60 cagctgcaga gcaagaaaac ttctggagcg acccaagcgg aaagagggtc acgcatcagg 120 tcgtctcacc atgcagagct tctcggcaca tcggccaatc aggaaaacaa ggcacaaagc 180 caatgcttgc aaaaagtctg ccagcttaag catgcaagcg ccagcaagag ctgggtggag 240 tcacgcgcac catcaaagta cagggcgctc tga 273 <210> 2 <211> 478 <212> DNA <213> Dendronephthya gigantea <400> 2 cccaaccccg atattccggt tggcaaggtg ctgggaaata ttgccagcag aggcgtcatg 60 ttcgcgatct gtctcactct ggacaacaga gaccacggat cactgaccct caatgttctc 120 cagggccagc agcaggagca caggaacatg ccggtggatg acgctatgtc attcatctct 180 aaacagtttc ccagcctcgt gggcggagga ggaggtggga actctgtgat atctcgggac 240 gggtcagcac tgccgggccc ctcccacacc ggccatcctc cagacatcag caagatactc 300 agcttcctca cagacgacag acctctctcc atcatggagt atgacaagat gattaaatac 360 ctggtcacca agagaacaga cactctgagg gaagagtacg gagacagcat cccagcccac 420 ctgcagcatc caccagtggg gcctcaccag gatccagcca ccaaggccaa gcaggaag 478 <210> 3 <211> 306 <212> DNA <213> Dendronephthya gigantea <400> 3 gggtgcctgc tggccgtttt gggctgcctc tccctcctgg cgggggcaca cggctacttt 60 atcaccgtgg acgcccatgc agaggaatgt ttcttcgaga aggtgacggc gggtacaaaa 120 ttaggcctgg catttgaggt tgctgaggga ggcttccttg acatagacat caagatctac 180 aaccctgaga tgaagaccat ccatgagggc gagcgcgagt ccaacgggcg gtacaccttc 240 cctgccagca tggacggcgt ctacacctac tgcttctcca acaagatgtc caccatgacg 300 cccaag 306 <210> 4 <211> 369 <212> DNA <213> Dendronephthya gigantea <400> 4 cttaaaattg acgacaagga ctacagcatt accttttcac tttatttcaa tgttcaatgg 60 tccgagccac gtttgaattt gtcccaagag tttttcaaca gtgaaaacat cactactgat 120 gagcagctgg tgccagtgaa tctggagctc attcatgacc tttgggtgcc aaacatttac 180 atatacaacc tgaaatcctt caaagtcatt gatgtcctct ctaaactggc tggtctatgg 240 attaacaaca agaaggaaat ctattatagc caagccactc acatcacctt catttgtcca 300 atgcttttcg actcgtttcc tctggatact caagtctgca agttccaagt tggcagctat 360 tcttacgac 369 <210> 5 <211> 366 <212> DNA <213> Dendronephthya gigantea <400> 5 aggtacaatg cccgcgggtt cgacatcaac cgcaacttcc cggactactt caagacgaac 60 aacaagcgat ctcagccgga gactgaggcg gtgaaggagt ggctgtccaa gatccagttc 120 gtcttgtcgg gcaacatcca cggcggcgcc ctggtggcct cctacccgtt cgacaacacg 180 ccaagctcca tcttcagctc ggtcctctcg tccccctcgc tcaccccgga cgacgacacc 240 ttcaaacacc tggccacaac ttactcactc aaccacggac gcatgtatct tggggacccc 300 tgcaaggttg gcgcgccaca gttcggaaac gggacgacga acggagcggc gtggtacccg 360 ctgact 366 <210> 6 <211> 423 <212> DNA <213> Dendronephthya gigantea <400> 6 atgtcaaagc gtggacgcgg tggtaaagga ggtggaaagt tccacatctc cctcgccctc 60 ccagttgctg ccgttatgaa ctgcgctgat aacactggcg ccaagtctct ctacgtcatc 120 gctgttgccg gcatcaaagg tcgattgaac cgactcccag ccgccggatc aggagatctc 180 gtcatggcct ccgtgaagaa aggaaagcca gaactccgaa agaaggtcca ccccgccgtc 240 gttgtccgac agtccaaggc tttccgaaga cgagacggta ccttcctgta cttcgaagat 300 aacgccggtg ttattgtcaa caacaaggga gaaatgaagg gctctgccgt cactggaccc 360 gtcgcgaagg aatgcgctga tctctggccc cgtatttctt ctaactcctc caccattgcc 420 taa 423 <210> 7 <211> 987 <212> DNA <213> Dendronephthya gigantea <400> 7 cttgtggctc tgacatgtct ctcacggtgg ctgtactggc aagtccacgc gatgtcacta 60 ggttgtgcgg tgggattgca aaggctgcgc tacacatgtg aagctatcgg ggccgagggc 120 tcaatccacg agtccatcga acagttgccg accttcgtgg ctggaccagc ccgacacggc 180 catggcctgg cgttggataa agacatgggc gtttcttaca gctcgttctt gggtgaggca 240 cctccgttga ggccacggca tgcggaaact gcgttcgacc agatgaccat ctacttcaag 300 ccacgcaagg aagcgctggt cgcccaggaa atgaaatgcg acgacgacat ggcttcttgc 360 tctcgcgaat gcgagcgctt atacggagac caagtttcga cgatgatgga accctgcaaa 420 gttgctgttg tggaaaagta tgctggtggc ggaggcggca gctgctttcc tgccaaggcc 480 gccgtctgga accagaacgt tggtcgcatc cgtatctccg acgtgcgcat cggtgacgag 540 cttgagactg gcagcggcaa ctcgtcgccg gttgtcgccc tactgcacgc tgatccggag 600 gcctgggtcg actacgtccg catctcgcat tctgccgggg aagacttgta catttcgcct 660 gcgcatatcc tgcagcttct ttctcgcagt caagggccca cctgggtgcc cgccagcctt 720 gttcgcccag gcgataactt gaagtcctca gcagggccga ccttggtgaa cgctgtggac 780 accgtgcggc tgaaaggcgc atacgcgccc ctgactgact cgggccagct gcttgtggag 840 ggcgtgctct gctcctgtta cgctcccccg cagagctttg agctctcaca caatatctgc 900 cactgggcca tgtttccgct gcgcgtcttt cacgctttga ggggtgtggt ggcgtccgca 960 gctcccgcac gcgttttcgt caccgca 987 <210> 8 <211> 1113 <212> DNA <213> Dendronephthya gigantea <400> 8 cccgtcctga tggaaaatct tcccacggac gcctgcctgc tcgttttcag gatcctcagt 60 gttctcggca tggcgatgcc caagattgcc agcattttcg tcctgggcat cgggactgcg 120 agaacttctc ggttgccaaa tggcgtcgcc gattacacgc taggcctcgt gcagctgcac 180 gcggagaaga ccgacggggc cctggctact gcgttccgca gtgaagatgc gccacagccc 240 gtggacgccg caagcagccc gattttggcg accgcggagg cggacaccaa atcgcagtgg 300 tgtttggctt cacaggcgaa ggcggtggag atgcatcagg aggtacagct gctcctggcg 360 cgaaatcgaa gcccgaacag taccgacgaa agccttatcc acatgaccta ctcgcagctc 420 caggaaggcc cgcagtctcg tatccttgac gaagagcaga tgcttgtgga tatggagtca 480 acggagttca ccgagaagtg cgatttgggc aacgaccttt ggttgctgaa ggacgaggtg 540 aatttcgctt cttgccctcc tgggaagtac ctccgtcatc agaccgcgga ggaccacaag 600 cgcaaacgga tgcgcctgcg gaaggccctg tgccagttga agctgcgcgg cgcacggcca 660 tggaacaagc gcgagcggtc gttcttcagg gcgcggggct actccacttt cgagtcgtcg 720 gtgtggccga agttgcccgg gtgcgcgtcc gtggtcgacg aaagctggta cagtgggcag 780 cacaagagct gggacatgcc cgccttccgc ccggacagcg cgcctccaca catgggcaag 840 ctggtccgcg ggaatctgga gttccacaag agctggaagg tggcatccac tggcttcccc 900 gactacttgc agtgcgagta tgacggcacc tggaaagagg ttccagtcac caaccccgtt 960 gggcagggct ccagtgtggt cttcgccgtg cgggagccta tcgggcgttg gatctccgct 1020 gtgggcgagc tgttggagcg atccatcaac cattggtgcc caaacgggcc ttgcagtaag 1080 caggacggtt tcgacctgat gacgacaatg gat 1113 <210> 9 <211> 372 <212> DNA <213> Dendronephthya gigantea <400> 9 ccacggattg ccctgttcgc tgtttgctcg gcgctcgtgg ttcagctggg catgtcgagc 60 tctcttcgaa cgcaacctgc agtgaaggtt gtggacgccc accagtgcaa agtgatgtgc 120 cagcgctttg cgtaccgctt catgggccct gagttccaag actccaagag ccctacggac 180 tgctccaaga agtgcgaggc agtttacggg ggcgcttcgc caacgagcgc cctggaggtc 240 cgtgcatgat gcgaatcagt gcaaggtgat ctgccagcgc ttcggcatgc cgatgcttgg tccggatttt 360 gaaggtatca ag 372 <210> 10 <211> 384 <212> DNA <213> Dendronephthya gigantea <400> 10 ggaggcgggt catgggagga ggactgggag gggatggtga ccagctccgt ggaggcggac 60 cccctggat acccttctct cctggtcacc tgacaccagc cacaccaggt acaagatgct ctacgcctcg 180 accaaggcca ccttcaagaa gcagttcggg gcggggcaga tcaaggacga atactacgcc 240 aacctgaagg aggaggtcac cctggcggga tacaagaagc acctgagtgt cgaggcggca 300 cctggccccc tgaccagggc ggaggaggag gccaaggaga tcaaggagaa ggagtcccgg 360 gtcgagatta gcgtggactc caag 384 <210> 11 <211> 341 <212> DNA <213> Dendronephthya gigantea <400> 11 gttaatactt ctcaaggagt accaatatct tgcattggga ttgcacaagt aaaaattgaa 60 ggaaagaatc aagatatgct tgctaatgca tgcatgcagt ttcttggaaa atcagaacat 120 gaaatccaac agattgcact tgaaactctt gaaggccatc aaagagcaat tatgggcaat 180 atgacagtgg aagaaattta ccaagacaga aagaaatttg cgaaagaggt gtttgaagtt 240 gcatcgtctg atcttctgca aatgggaata tatgttgtat catacacact gaaagatgtg 300 acagacaacg aaggatacct tgcagccctt ggaaaaaccc g 341 <210> 12 <211> 372 <212> DNA <213> Dendronephthya gigantea <400> 12 atggccatgg caatgcagaa aaaattgaat gttcgattac ccccagaagt caatcgcata 60 ctttacgtcc ggaatctacc atacaagatt acatcagaag aaatgtatga tatattcggg 120 aaatatggag ccatacgaca aatccgtgtt ggaaacacag cagaaacaag aggtacagct 180 tttgtggtat atgaagatat ttttgatgca aagaatgctt gtgaccacct ttcgggtttt 240 aatgtttgca accgctattt agtagttctt tactatcagc ccacaaaagc cttcaagaag 300 gtagacacag ataaaaagaa agaagaaatc gagaaaatga aggccaaatt tgggttgaat 360 gaagatgaat aa 372 <210> 13 <211> 405 <212> DNA <213> Dendronephthya gigantea <400> 13 atgctgcgaa ctgctgttct cgcatttatc tgcagttgct tggtccagac gacgtacggg 60 gggaccgtcg aagcgaagtt cctttcccgc ggccaacgca gtgaagacga ggtgaagcac 120 gacaccgcgc gccaatacca gtcagtggtc aggcttcacg acaatgctga gaggaggctg 180 accagaatcg tcgtcgagtt gttgggtgtc gacgagtctg aggtcaagcg tgatgcgagc 240 ttcgtggaag accttggcgc tgatgagctg gacacggtgg aactcgtcgt ggcagtgggg 300 gaggagtttg gcttccaaat tccagatgat gacgcggagt cgttggagac atttggagac 360 ttggtcgact acgtgaataa ttacgaacac aacacagtcc gttga 405 <210> 14 <211> 270 <212> DNA <213> Dendronephthya gigantea <400> 14 ggaggactca gccgggaaga tatcatatct ttagccactt caggacaatc gcttggtggc 60 gacatgagtg agctggacga tgctggaact tactttgatc tgggattgcg caaagtgaca 120 atgccgggaa cttatcaata catgtgcacc cggaacaaca acttctcgaa ccggagtcag 180 aaaggaaagc tggtggtcac caactccgat ttcagacaac agagcgttga ccaaaatggc 240 ggcgtcgtgg tcgtctccga cgtcgagtaa 270 <210> 15 <211> 290 <212> DNA <213> Dendronephthya gigantea <400> 15 atggaacgca gcgagagcaa ccagttggag gctgcacctg tcctttgccg ggcgggctgc 60 ggcttctttg gctcctcgaa cactgaagga ctatgttcaa aatgttacaa agaccaattg 120 aagaaaaagc agcaaccccc aaatcaaagc aataatactt cttcatccac tgcaacagca 180 cagccaacag ttccttctat cacaccgcaa ataagcgaaa gctcatccac atctcccacc 240 acatcctcta caactgcatc ggttgccaca tcttcagtag cagtgccaag 290 <210> 16 <211> 426 <212> DNA <213> Dendronephthya gigantea <400> 16 tggagttatt ataattaccc gttggttgtc ggtatgtcta catctctact gaaaaagagt 60 ttagaactct ttgatgaaga gggaaagacg gacagaacca aatcaaacaa aggacgccct 120 gctcgtacca ttgtgaagaa taaacaagcc actctgaaag ataggaaagt gaagtcagca 180 ttagaatcat acacgaagaa gaaccgtaag gaagacagga cacttggaaa tttggctgtt 240 cttgaaaaga tatcgctgaa aaactctgtt agccaagact cggccaacaa aattctgaaa 300 tatcatctgc agaagtctcg gaaagttgtg gaagctgagc caaagaaaga ggccgagaag 360 tctgtgttta ctgatgaaga tttccgaaag ttccagcagg aatattttgt caaaggaaag 420 atataa 426 <210> 17 <211> 1107 <212> DNA <213> Dendronephthya gigantea <400> 17 atgaacggct acggtgtgac cgccgagcag cggtctttgt tgaacagaaa cagtcacagc 60 ttgaaaccac tatccgctgc cggccacctc aacatctggc aggtggtcgt gcaagccatc 120 gtaccgatcc tcatcttctt gttcgcgctc gggatcctga caatcacgcc acgctattcc 180 agccctgact ccgtgtccac gctctgcgcc attggccttt tcggcgcttt gctgtacccc 240 acgatctcct tcctgctggt ggtcgtaggg gttgtgtcaa cgaagatgcg taccagaata 300 cgctggcccc cagtcgtctt ctggtcatgg tggccgtgct ggaagttctc cgcctgctgt 360 ctcgcggcgt ttctgggcat cagcatcggc aactacatct ggtacaagca gctcctgccc 420 taccaccaga tggaccgcct ccaggcatat gacaatgtca acccttccga ggtgaccggc 480 actcggatgc aggacgccgg ggtcgtcgag ttcaagtcca ccaacggtgt ggatcgcaca 540 cggactgggt gcatcaagaa tggtgccacc tactgcatcg cccctgtcgt gctgaatggc 600 aaggtggagc caggtgtggc accaggggag acgtacgact tcttcatggc agggaaggac 660 tgctgcagct gccccggcga gttccgttgt ggcgactgga acatgccgtc gccaactcta 720 ggtgggctgc gaatcgtgga ctcgggcgat cgcgccttct acaggctcgc cgcggagcag 780 tggggcagcc tgtacggcaa gcccgtggag cacccgctgt tcttcacgtg gtcaacggat 840 ccggtggaga cctggcacga aatgcgcacg acagcgacac gtgaggcctg tctggccatg 900 ctcgtggtcc catttttctt gatcacgtac gccttcctcg ctaatggtct gttgggcatc 960 ctccacgatc ggggctacgc ggcgccccaa gaggctccga tgccgcctgc gggcatgggg 1020 cgcgacatga ctgcgaagtt cctgccccag atgcacaagt acacggcaga ctcgcaggct 1080 cagcaggccg gcgcgaccgt gatctga 1107 <210> 18 <211> 1821 <212> DNA <213> Dendronephthya gigantea <400> 18 atgtcggcaa cccctgcgcc gatcgtggag ggctgcccgg cgttctcgga aggatgcccg 60 ttctccaagc tcgacatcgc gctgctgcca caggtcatcc aggaggtgcc gaaggagatc 120 acggagaagt gccctgcatt tgagaagggc tgccctttca aggaggacga ctccgtggag 180 gcgctgtaca agcgcatgtc ggaaatgccg gcctcgcacc gcatgggcca ggagagcccc 240 tcacccgcgg cacagaaggt ggaggccacg ctgcgcctgg tgcacgagca gtcgaaggcc 300 ttgaagtcca ggttgaaagc cacctgcccc gtcttcgcca catcctgccc tttcaagaca 360 gtaacctcgg acggccagcc cttggtgcac gagttggacg acgtcgtcga gaggtgggga 420 ctgaaggaga tcgaggaggt gatcacctct cctgtgtcag cgaacgccga gccgctctcc 480 aagaccatga aagctgggac caggtctgtg caccgcgctg ccgagaacgt ccgatttgtc 540 cgcgacttcc tcaagggcac ggtgcccaaa gacagctacg tggagctgtt gcgtgcgttg 600 taccatgtct acgacgccct ggagcgagct atccgaggcc tgccagagca tttgcagcac 660 tgcgacttca acaagttgtg gcgcaccgag accttgcttg ccgacctgtg ccactacacc 720 aacagcgcct acccagagac cccggaggct gtgagcagaa tcgtcggtgt tccttctcag 780 gtggcccagc agtacgtgga tcatttggac gctctcgggg agcaggagcc cctgctggtg 840 ttggcgcacg cctacacccg ctacctgggg gacttgagcg gcggccagat cctggcccga 900 gcggctgaga aggcgtacgg cctggagggc ggccgaggca ctgcattcta cagcttcgag 960 ctggtgggaa cagctgcgtc cgaggtcaag gacttcaaga aagcgtacag agcgtccttg 1020 gacgcgttgc agctgaaagc acacgaggcg gacgcgttgg tggaggaggc caacatggct 1080 ttcttgatga acatgctcct cttcgaggac cgggatgtcg ctgctggtca cctagcgcga 1140 gtgcggactc tggaagaggc tcgcgagctg gtggcagcga acaagagtgc gttgaagttc 1200 cagcaggcct acgctggggg tagcaccgca gcagggaagt gccccttctt gccgccgcca 1260 gagcagcggg gggccagtgg cgccagctgc ccctggccct tcttgtggct gcacgacccc 1320 cgcgcagcct tgatggggca tccctacaaa aatctcaccg gtgctctcgt ggcagcgggc 1380 ttcacgaagt ttgcatggga gcgtcctcgc agtgcagcgg tcgggtcttt gttgtgtgcc 1440 gtgggctgct acagcttgaa gccgacgcgc aatacccaag ccgggcgtgg cgtctgcccg 1500 ttcatgcctg catccacgaa cagcgcgtcg ccggcatcgc ggagcgccgc ccaagaacac 1560 catggcgacc gtgtgtgccc atggcctttc gtgtgcttcc acgacccccg tgctgcgttc 1620 cggggccacc cttgcaagaa cgcgtgcggt gtgctggcga cggcgggctt tgcgaaggtc 1680 gcatgggagt accctcgcag cgctgcggcc gggtcgctga tctgtgcgct ggggtgctac 1740 agtctgaagc caaagcgcaa gagcatttcg aatgatgcgg cgccagcctg tgaacctgcc 1800 gaccggatgc ttgccgcctg a 1821 <210> 19 <211> 204 <212> DNA <213> Dendronephthya gigantea <400> 19 tccgacagtt ccctcaatga tttcctccat gcggactccg tcagcatagc ttccagctgg 60 ccctgcccca gatccacggg tgcgcccatt ggtatcctgg aaggctctga tcagcagatg 120 caccagccta ggaatggatc cattctcacg gagcggcgca tgattggccg ggcacagagc 180 caagttcctg atcaacccaa taac 204 <210> 20 <211> 20 <212> DNA <213> Dendronephthya gigantea <400> 20 agatgaagca taaccctcaa 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 21 agatgaagca taaccctcaa 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 22 cttaagctgg cagacttttt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 23 aaacaagcca ctctgaaaga 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 24 ctttcggaaa tcttcatcag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 25 agtcacagct tgaaaccact 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 26 cgtattctgg tacgcatctt 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 27 cagaggaatg tttcttcgag 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 28 ttgttggaga agcagtaggt 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 29 acgacaagga ctacagcatt 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 30 ccatagacca gccagtttag 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 31 aacaagagtg cgttgaagtt 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 32 tgagattttt gtagggatgc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 33 cggactactt caagacgaac 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 34 ccgtggttga gtgagtaagt 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 35 aagaaaggaa agccagaact 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 36 tggtggagga gttagaagaa 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 37 cgtttcttac agctcgttct 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 38 acttttccac aacagcaact 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 39 ctactccact ttcgagtcgt 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 40 ctcttgtgga actccagatt 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 41 acagttccct caatgatttc 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 42 gttattgggt tgatcaggaa 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 43 atcactgacc ctcaatgttc 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 44 ttccctcaga gtgtctgttc 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 45 ctgagttcca agactccaag 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 46 agatcacctt gcactgattc 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 47 acaccaggta caagatgctc 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 48 ctccttctcc ttgatctcct 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 49 aatatcttgc attgggattg 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 50 cgcaaatttc tttctgtctt 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 51 acacagcaga aacaagaggt 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 52 catcttcatt caacccaaat 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 53 aataccagtc agtggtcagg 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 54 aagtctccaa atgtctccaa 20 <210> 55 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 55 atcaaagcgc aggagaag 18 <210> 56 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 56 gaagtccacc ctttcagtg 19 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 57 tttagccact tcaggacaat 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 58 gttgtctgaa atcggagttg 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 59 cctcgaacac tgaaggacta 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotides <400> 60 ggcactgcta ctgaagatgt 20

Claims

A microarray for confirming whether or not the oligonucleotide of the nucleic acid sequence of any one or more genes selected from the following group or the complementary strand molecule thereof is integrated into acidified sea water:
(L-Asparaginase), a gene described in SEQ ID NO: 3 (Cadherin EGF LAG seven-pass G-type receptor), SEQ ID NO: 4 (DNA-directed RNA polymerase III subunit), the gene described in SEQ ID NO: 6 (Dynactin subunit 1), the gene described in SEQ ID NO: 7 (FAD -dependent oxidoreductase), Ficolin (SEQ ID NO: 8), Myosin light polypeptide 6, Phosphoenolpyruvate carboxykinase (SEQ ID NO: 10), Gene A polyketide synthase, a gene described in SEQ ID NO: 13 (Protein phosphatase 1 regulatory subunit 12A), a gene described in SEQ ID NO: 14 (Pyrroline-5-carboxylate re the gene described in SEQ ID NO: 16 (Alcohol dehydrogenase 1), the gene described in SEQ ID NO: 17 (Aquaporin TIP2-3), the gene described in SEQ ID NO: 18 A gene described in SEQ ID NO: 19 (Guanidinoacetate N-methyltransferase) or a gene described in SEQ ID NO: 20 (Protein tyrosine phosphatase type IV A3).

2. The method according to claim 1, wherein the gene is selected from the group consisting of a large resin beetle ( Dendronephthya gigantea. &lt; / RTI > The microarray for detecting exposure to acidified sea water.

The microarray for confirming exposure to acidified sea water according to claim 1, wherein the acidified sea water has a pH of 6 to 7.7.

1) isolating the RNA from the large resin beetle, which is an experimental group exposed to acidified sea water, and the large resin beetle, which is a control group;
2) labeling the experimental group and the control group with different fluorescent substances while synthesizing cDNA from the RNA of the experimental group and the control group of step 1);
3) hybridizing the cDNA labeled with each of the different fluorescent substances of step 2) with the microarray of claim 1;
4) analyzing the reacted microarray; And
5) checking the degree of gene expression integrated in the microarray of claim 1 against the control group in the analyzed data to confirm whether the acidified sea water is exposed.

The method according to claim 4, wherein the fluorescent material of step 2) is selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (RITC), rhodamine-B-isothiocyanate (RITC), and rhodamine To determine whether the acidified sea water is exposed.

1) isolating the RNA from the large resin beetle, which is an experimental group exposed to acidified sea water, and the large resin beetle, which is a control group;
2) Performing Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a primer pair complementary to the following genes and capable of gene amplification, using the RNA of step 1) as a template,
(L-Asparaginase), a gene described in SEQ ID NO: 3 (Cadherin EGF LAG seven-pass G-type receptor), SEQ ID NO: 4 (DNA-directed RNA polymerase III subunit), the gene described in SEQ ID NO: 6 (Dynactin subunit 1), the gene described in SEQ ID NO: 7 (FAD -dependent oxidoreductase), Ficolin (SEQ ID NO: 8), Myosin light polypeptide 6, Phosphoenolpyruvate carboxykinase (SEQ ID NO: 10), Gene A polyketide synthase, a gene described in SEQ ID NO: 13 (Protein phosphatase 1 regulatory subunit 12A), a gene described in SEQ ID NO: 14 (Pyrroline-5-carboxylate re the gene described in SEQ ID NO: 16 (Alcohol dehydrogenase 1), the gene described in SEQ ID NO: 17 (Aquaporin TIP2-3), the gene described in SEQ ID NO: 18 A gene described in SEQ ID NO: 19 (Guanidinoacetate N-methyltransferase) or a gene described in SEQ ID NO: 20 (Protein tyrosine phosphatase type IV A3); And
3) comparing the gene product of step 2) with the control group to confirm the degree of expression.

A kit for confirming exposure to acidified sea water, comprising the microarray of claim 1.

8. The kit according to claim 7, wherein the acidified sea water has a pH of 6 to 7.7.

The method according to claim 7, further comprising adding any one selected from the group consisting of streptavidin-like phosphatase conjugate, chemifluorescent, and chemiluminescent fluorescent substance Wherein the acidicized sea water is exposed to atmospheric air.

The method according to claim 7, wherein the buffer solution is selected from the group consisting of buffer solution used for hybridization, reverse transcriptase for synthesis of cDNA from RNA, dNTP and rNTP (premixed or separated feed type), labeling reagent, and washing buffer solution Wherein the acidic aqueous solution is added to the aqueous solution.

Kit for confirming exposure to acidified sea water containing a pair of primers complementary to each of the following genes and capable of amplifying the gene:
(L-Asparaginase), a gene described in SEQ ID NO: 3 (Cadherin EGF LAG seven-pass G-type receptor), SEQ ID NO: 4 (DNA-directed RNA polymerase III subunit), the gene described in SEQ ID NO: 6 (Dynactin subunit 1), the gene described in SEQ ID NO: 7 (FAD -dependent oxidoreductase), Ficolin (SEQ ID NO: 8), Myosin light polypeptide 6, Phosphoenolpyruvate carboxykinase (SEQ ID NO: 10), Gene A polyketide synthase, a gene described in SEQ ID NO: 13 (Protein phosphatase 1 regulatory subunit 12A), a gene described in SEQ ID NO: 14 (Pyrroline-5-carboxylate re the gene described in SEQ ID NO: 16 (Alcohol dehydrogenase 1), the gene described in SEQ ID NO: 17 (Aquaporin TIP2-3), the gene described in SEQ ID NO: 18 A gene described in SEQ ID NO: 19 (Guanidinoacetate N-methyltransferase) or a gene described in SEQ ID NO: 20 (Protein tyrosine phosphatase type IV A3).

12. The kit according to claim 11, wherein the acidified sea water has a pH of 6 to 7.7.