JP2008000131A - Method for evaluating physiological state of test aquatic organisms - Google Patents

Abstract

The present invention provides a method for confirming the physiological state of a test aquatic organism by gene expression and evaluating whether or not the test organism is suitable as an indicator animal of a standard state in a biological test. Is an issue. In addition, the physiological state of test aquatic organisms under various breeding conditions is confirmed by gene expression, and it is evaluated whether or not the breeding conditions are suitable as conditions for breeding an indicator organism in a standard state in a biological test. It is also an object to provide a method for doing this.
[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors conducted comprehensive gene expression analysis on test aquatic organisms and identified genes of aquatic organisms involved in physiological states. As a result, it was found that the medaka gene group involved in the physiological state was identified and the physiological state of the test organism could be confirmed by confirming the expression of this gene group.
[Selection figure] None

Description

  The present invention relates to a method for confirming the physiological state of a test aquatic organism by gene expression and evaluating whether or not the test organism is suitable as an indicator animal of a standard state in a biological test.

  Currently, various indicator organisms are used in biological tests. The indicator organisms used in biological tests are acclimated in advance and undergo visual inspections such as low mortality, no illness, no behavioral abnormalities, and good health. It is used for the test only after it is judged that the breeding method during acclimatization is appropriate by confirming that the indicator organism is in a standard state by an external inspection method. Furthermore, at the end of the biological test, it is judged that the breeding method under test is appropriate by confirming that the indicator organism in the control group is in the standard state by visual inspection, and the test is considered to be effective. . In addition, even in the case of biological investigation in the field, the physiological state of the indicator organism is evaluated by visual inspection.

  However, even if it is recognized that there is no problem in the appearance inspection, it has been pointed out that there is a difference in the physiological state, and it is difficult to reliably evaluate the physiological state of the indicator organism only by the appearance inspection. Yes. Differences in the physiological state of the indicator organism at the time of acclimatization are considered to cause differences in subsequent biological tests. In addition, since there is a difference in the physiological state of the control group at the end of the biological test, there is a current situation in which the result of the biological test cannot be accurately determined.

  That is, it is suggested that there is a risk that an accurate test result cannot be obtained because only the appearance inspection has a difference in the physiological state of the control group before or during the test of the indicator organism used in the test. Therefore, it is required to establish a method for objectively evaluating not only the physiological state of an indicator organism by a tester's subjective judgment of visual inspection but also by a scientific method.

  Medaka is an aquatic species recommended for the ecotoxicity test required by the “Amendment of Chemical Examination Law (Law Concerning the Examination and Regulation of Chemical Substances)” (Japan) enforced in FY2004. Medaka's ecotoxicity tests are to be conducted by GLP-certified institutions. Although a detailed medaka breeding method is provided as a standard manual, there is no method for objectively evaluating whether a medaka bred by this manual is in a standard state.

  In recent years, comparative analysis methods between the control group and the test group after it has been determined that the biological tests conducted are effective are not only morphological and histological methods, but also physiological and biochemical methods, Various analysis methods, such as measurement of gene expression and protein expression status, are used, and the physiological state of an organism can be analyzed in detail. Among these, it is known that the comprehensive measurement of gene expression status can easily determine the physiological state of an organism.

  However, the technology for measuring the comprehensive gene expression status has been used in evaluating whether test organisms (eg test aquatic organisms) are suitable as standard state indicator organisms in biological tests, Never before.

Prior art document information related to the invention of the present application is shown below.
OECD Guidelines for Testing of Chemicals, "Fish, acute toxicity test" Vol. 203, 1-9, 1992 USEPA, EPA / 600 / 4-90-027F, "Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, 4th ed.", (1993) Amended Chemical Substances Control Law (Ministry of Health, Labor and Welfare, Ministry of Economy, Trade and Industry, Ministry of the Environment, Japan, February 2004)

  The present invention has been made in view of such circumstances, and its purpose is to confirm the physiological state of a test aquatic organism by gene expression, and the test organism is an indicator organism of a standard state in a biological test. It is in providing the method of evaluating whether it is suitable as.

  That is, rather than assessing the physiological status of the indicator aquatic organisms by visual inspection, the physiological status of the test aquatic organism planned to be used as the indicator organism is measured by measuring the gene expression status, followed by a biological test It is an object to provide a method for evaluating whether or not it is suitable. Alternatively, an object of the present invention is to provide a method for examining the physiological state of a test aquatic organism to be used as a control group indicator organism during a test by measuring the gene expression status and evaluating the effectiveness of the test.

  In addition, the physiological state of test aquatic organisms under various breeding conditions is confirmed by gene expression, and it is evaluated whether or not the breeding conditions are suitable as conditions for breeding an indicator organism in a standard state in a biological test. It is also an object to provide a method for doing this. Furthermore, there is also provided a method for growing an indicator organism in a standard state in a biological test under the breeding conditions specified by the evaluation method, and provision of an aquatic organism that can be used as an indicator organism in a standard state grown by the breeding method. Let it be an issue.

  In order to solve the above-mentioned problems, the present inventors have made use of an aquatic organism (medaka) to be used as a standard indicator organism for biological tests under various breeding conditions, or a control group under biological tests. We conducted a comprehensive gene expression analysis for the indicator aquatic organisms (medaka) to be used as the target, and identified the aquatic organism genes involved in the physiological state.

  Specifically, we studied the physiological state of medakas acclimated for ecotoxicity tests. First, the Ecotoxicity Test Standard Manual was verified, and it became clear that the gene expression status of mature medakas acclimated under the same conditions according to the standard manual was very stable, although there were gender differences in males and females. (Figure 1a, 1b).

  Next, we examined the effects of breeding with different diets on the physiological state of medaka. As a result, it was clarified that the kind of feed to be given greatly affects the survival rate and growth state of medaka (FIG. 2). In addition, it is clear that even if acclimation is performed in the same environment, the gene expression status of mature medaka varies greatly depending on the feed, and the expression status of each gene correlates with the survival rate and growth status of medaka. (Figure 1c).

  Next, we examined how the low-dissolved oxygen treatment affects the physiological state of the control medaka in the ecotoxicity test. As a result, it was clarified that the dissolved oxygen concentration greatly affects the survival rate and growth state of medaka. In addition, it is clear that the gene expression status of mature medaka varies greatly depending on the dissolved oxygen concentration even when acclimated in the same environment, and the expression status of each gene correlates with the survival rate and growth status of medaka (Fig. 1d).

  Furthermore, when the physiological state of medaka deteriorated, that is, when the survival rate and the growth state deteriorated, genes whose expression levels were greatly changed were identified. As a result, from 26,689 types of genes, 23 types (SEQ ID NOs: 1 to 23) of genes whose expression levels were changed by 1/4 times or less and 93 types of genes whose expression levels were changed by 1/2 times or less (SEQ ID NOs: 1 to 93), 18 types (SEQ ID NOs: 93 to 111) of genes whose expression levels were changed by 2 times or more were identified (Table 1).

  In addition, 27 types (SEQ ID NOs: 112 to 138) of genes whose expression level of the gene did not change were identified as negative controls even in an environment that is thought to affect the physiological state of medaka (Table 2). As an example of the usage method of this negative control, the expression level of the gene used for the evaluation of the physiological state and the negative control gene is determined for the test aquatic organism and the medaka bred in the normal control group. At that time, the expression level of the negative control gene is considered not to change between the test aquatic organism and the control group. That is, it is possible to correct the expression level of the gene used for the evaluation of the physiological state from the expression level of the negative control gene. Further, the expression level of the negative control gene itself may be measured and compared between the test aquatic organism and the normal control group. If there is a difference in the expression level, it is a measurement error, noise, or a subtle difference in the implementation environment, and can be used to correct actual measurement values.

  Furthermore, when the gene expression state of each medaka bred under different dissolved oxygen conditions was confirmed by DNA microarray analysis, the breeding under high dissolved oxygen conditions deteriorated the physiological state of medaka. It became clear. It became clear that the physiological state of medaka can be evaluated regardless of the survival rate by using the aquatic organism genes involved in the physiological state identified in the present invention.

  That is, the present inventors have found that the physiological state of a test organism can be confirmed by identifying a medaka gene group involved in the physiological state and confirming the expression of this gene group. In addition, by applying this method, it is possible to evaluate whether or not the test organism is suitable as an indicator animal in a standard state in a biological test, and breeding an indicator organism in a standard condition in a biological test. The present inventors have found that it is possible to evaluate whether or not it is suitable as a condition for doing so, and thereby completed the present invention.

More specifically, the present invention provides the following [1] to [16].
[1] A method for evaluating the physiological state of a test aquatic organism, comprising the following steps (a) to (c).
(A) a step of determining an expression level of aquatic organism genes involved in a physiological state in a biological sample derived from a test aquatic organism (b) an expression level of the gene in a biological sample derived from a test aquatic organism The step of comparing the normal control expression level of the gene (c) The step of evaluating whether or not the physiological condition of the test aquatic organism is good based on the comparison result of the expression level in (b) [2] The involved aquatic organism gene is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 23, and the expression level of the gene group in the test aquatic organism is a normal control The method according to claim 1, wherein when the expression level is lower than the expression level, it is evaluated that the physiological state of the test aquatic organism is not good.
[3] The aquatic organism gene involved in the physiological state is a gene group consisting of at least one or more base sequences selected from the base sequences set forth in SEQ ID NOs: 1 to 93, and the gene group in the test aquatic organism The method according to claim 1, wherein the physiological state of the test aquatic organism is evaluated to be poor when the expression level of is lower than the normal control expression level.
[4] A gene group consisting of at least one or more base sequences selected from the base sequences set forth in SEQ ID NOs: 93 to 111 as the aquatic organism genes involved in the physiological state, the gene groups in the test aquatic organism The method according to claim 1, wherein the physiological state of the test aquatic organism is evaluated to be poor when the expression level of is higher than the normal control expression level.
[5] A test aquatic organism comprising the following steps (a) to (c) is suitable as an indicator organism in a standard state in a biological test, or an indicator organism serving as a control group in a biological test. How to evaluate whether or not.
(A) a step of determining an expression level of aquatic organism genes involved in a physiological state in a biological sample derived from a test aquatic organism (b) an expression level of the gene in a biological sample derived from a test aquatic organism According to the comparison result of the expression level in the steps (c) and (b) of comparing the normal control expression level of the gene, the test varicella organism is Step [6] for evaluating whether or not it is suitable as an indicator organism to be used as a control group At least one or more base sequences selected from the base sequences described in SEQ ID NOs: 1 to 23 as the Minamata organism genes involved in the physiological state When the expression level of the gene group in the test aquatic organism is lower than the normal control expression level, the standard indicator organism in the biological test, or biology The method according to claim 5, wherein the method is evaluated as unsuitable as an indicator organism serving as a control group during a physical test.
[7] The aquatic organism gene involved in the physiological state is a gene group consisting of at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 93, and the gene group in the test aquatic organism 6. When the expression level of is lower than the normal control expression level, it is evaluated that it is not suitable as an indicator organism in a standard state in a biological test or as a reference indicator organism in a biological test. The method described in 1.
[8] A gene group consisting of at least one or more base sequences selected from the base sequences set forth in SEQ ID NOs: 93 to 111 as the aquatic organism genes involved in the physiological state, the gene groups in the test aquatic organism 6. When the expression level of is higher than the normal control expression level, it is evaluated that it is not suitable as a standard indicator organism in a biological test or a control indicator organism in a biological test. The method described in 1.
[9] A method for evaluating whether the test breeding conditions including the following steps (a) to (c) are suitable as conditions for breeding an indicator organism in a standard state in a biological test.
(A) a step of determining the expression level of aquatic organism genes involved in physiological conditions in a biological sample derived from aquatic organisms cultivated under test breeding conditions (b) biology derived from aquatic organisms to be examined The comparison of the expression level of the gene in the experimental sample with the normal control expression level of the gene (c) (b) shows that the test breeding condition is an indicator organism of the standard state in the biological test. Step [10] for evaluating whether or not it is suitable as a condition for breeding at least one base sequence selected from the base sequences described in SEQ ID NOs: 1 to 23 as the Minamata organism gene involved in the physiological state When the expression level of the gene group in the test aquatic organism is lower than the normal control expression level, the test breeding condition is The method according to claim 9, wherein the method is evaluated as unsuitable as a condition for rearing an object.
[11] A gene group consisting of at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 93, wherein the aquatic organism gene involved in the physiological condition is the gene group in the test aquatic organism The test breeding condition is evaluated as not suitable as a condition for breeding an indicator organism in a standard state in a biological test when the expression level of is lower than the normal control expression level. Method.
[12] A gene group consisting of at least one or more base sequences selected from the base sequences set forth in SEQ ID NOs: 93 to 111 as the aquatic organism genes involved in the physiological state, the gene groups in the test aquatic organism The test breeding condition is evaluated as not suitable as a condition for breeding a standard state indicator organism in a biological test when the expression level of is increased from the normal control expression level. Method.
[13] According to the method according to any one of claims 9 to 12, aquatic organisms are obtained under test breeding conditions evaluated as being suitable as conditions for breeding indicator organisms in a standard state in biological tests. How to train.
[14] The aquatic organism grown according to claim 13, which can be used as an indicator organism in a standard state in a biological test.
[15] The gene group consisting of at least one or more base sequences selected from the base sequences set forth in SEQ ID NOs: 112 to 138 is used as a negative control. A method for evaluating a physiological condition of a test aquatic organism, a physiological condition of an indicator organism, or a breeding condition of a test aquatic organism / indicator organism.
[16] A nucleic acid microarray comprising one or more probes corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 1 to 138.

  In the present invention, genes involved in the physiological state of aquatic organisms have been identified, and it has become possible to easily evaluate the physiological state of test aquatic organisms using this gene as an index. By using this evaluation method, it is considered that a difference in physiological state in the indicator organism that could not be detected by a conventional visual inspection performed before the biological test can be confirmed. Therefore, when a biological test is conducted using aquatic organisms that are evaluated to be in the standard state by this evaluation method, the results are considered to be more accurate. In addition, by evaluating the physiological state of the control aquatic organisms in the ecotoxicity test using this evaluation method, the results of the ecotoxicity test will be more accurate.

  Further, by applying such an evaluation method, it is possible to assess the habitat or breeding method of the indicator organism. In this case, the indicator organism is not limited to an organism bred in an experimental facility, but may be an organism living in the field.

  The present invention is based on the discovery of gene expression patterns associated with the physiological state of aquatic organisms. In the present invention, aquatic organisms are not particularly limited, but preferably include fish, more preferably medaka.

  The present invention is characterized by a method for evaluating the physiological state of a test aquatic organism by determining the expression level of aquatic organism genes involved in the physiological state in a biological sample derived from the test aquatic organism.

  The differentially identified gene identified herein is used as a marker for evaluating the physiological state of a test aquatic organism or as a gene target whose expression is to be maintained in order to improve the physiological state.

  Genes whose expression levels are modulated in aquatic organisms grown under other environmental conditions compared to normal control aquatic organisms (Tables 1 and 2 in the Examples) are collectively referred to herein as “physiology. It is referred to as “aquatic organism genes involved in the state”, and the corresponding encoded polypeptide is referred to as “aquatic organism protein involved in the physiological state”. Unless otherwise specified, “aquatic organism gene involved in physiological condition” refers to any sequence disclosed herein (for example, the sequence described in any one of SEQ ID NOs: 1 to 138). means. The sequence number of the gene and the accession number of the database are shown in Table 1 and Table 2 in the Examples.

  The physiological state of the test aquatic organism is evaluated by measuring the expression of various genes in the biological sample derived from the test aquatic organism. Similarly, by measuring the expression of these genes in response to various environments, it is possible to evaluate the optimum environment as the growth conditions for the test aquatic organisms.

The present invention relates to a method for evaluating the physiological state of a test aquatic organism, including the following steps (a) to (c).
(A) a step of determining an expression level of aquatic organism genes involved in a physiological state in a biological sample derived from a test aquatic organism (b) an expression level of the gene in a biological sample derived from a test aquatic organism The step of comparing the normal control expression level of the gene (c) and the step of evaluating whether or not the physiological condition of the test aquatic organism is good based on the comparison result of the expression level in (b)

  As the first step of this method, the expression level of at least one of the aquatic organism genes involved in the physiological state is determined in a biological sample derived from the subject aquatic organism.

  As the biological sample derived from the test aquatic organism, when the aquatic organism is small, a cell sample obtained from the whole aquatic organism may be used. When the aquatic organism is large, a part cell sample of the aquatic organism may be isolated and used as a biological sample. Gene expression may also be measured from other body fluids such as the blood of a test aquatic organism. Other biological samples can be used to measure protein levels.

  The gene group for determining the expression level is not particularly limited, but preferably a gene group consisting of one or a plurality of base sequences of the sequences set forth in any one of SEQ ID NOs: 1-111 is more preferable. Can include a gene group consisting of one or a plurality of base sequences of the sequences described in any of SEQ ID NOs: 1 to 23.

  The expression level of aquatic organism genes involved in the physiological condition in a specific specimen is the mRNA corresponding to the base sequence described in any of SEQ ID NOs: 1 to 138 or the base described in any of SEQ ID NOs: 1 to 138. It can be determined by quantifying the protein encoded by the sequence. Methods for quantifying mRNA are known to those skilled in the art. For example, the level of mRNA corresponding to the base sequence described in any one of SEQ ID NOs: 1 to 138 can be estimated by Northern blotting or RT-PCR. A person skilled in the art can design a nucleotide sequence of a probe or primer for quantifying the base sequence described in any one of SEQ ID NOs: 1 to 138. The probe may contain at least 10, 20, 50, 100 nucleotides of “aquatic organism genes involved in physiological state”. In addition, the expression of the gene can be measured using a PCR assay utilizing reverse transcription using a primer specific for a sequence having a difference in expression.

  The expression level of the gene containing the base sequence described in any one of SEQ ID NOs: 1 to 138 may be analyzed based on the activity or expression level of the protein encoded by the gene. A method for determining the amount of the protein encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 138 is well known in the art. For example, immunoassay methods are useful for the determination of proteins in biological materials. Any biological material can be used for the determination of the protein or its activity. The biological activity of the protein encoded by the gene can also be measured by methods well known in the art.

  In the present invention, it may be used as a determination reagent for evaluating the physiological state of aquatic organisms. The determination reagent here includes a compound that binds to the polynucleotide or polypeptide of the present invention. Preferably, an antibody that binds to an oligonucleotide that hybridizes with the base sequence described in any of SEQ ID NOs: (1 to 138) or a protein encoded by the base sequence described in any of SEQ ID NOs: (1 to 138) Can be used as such compounds.

As a second step of the method, the expression level of one or more aquatic organism genes involved in the physiological state in the biological sample of the test aquatic organism is compared to the normal control level of the gene. In the present invention, the “normal control level” means an international toxicity test standard manual (for example, OECD Guidelines for Testing of Chemicals, “Fish, acute toxicity test” Vol. 203, 1-9, 1992; 2) USEPA, EPA / 600 / 4-90-027F, "Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, 4 ^th ed.", (1993) Manual specified in the Ministry of Labor, Ministry of Economy, Trade and Industry, Ministry of the Environment, Japan (February 2004)) of genes in biological samples derived from aquatic organisms bred or acclimated according to the procedures and conditions specified This refers to the expression level, and the fluctuation range of the gene expression level is within the allowable range of the accumulated data.

  Specific examples of breeding conditions specified in the International Toxicology Test Standard Manual of the Chemical Substances Control Law include the following. Water temperature: constant at 21-25 ° C, fluctuation is ± 1.0 ° C, dissolved oxygen concentration in water: at least 80% of saturated oxygen concentration, light-dark cycle: 12-16 hours light (test conditions are 16 hours), feeding: Brine shrimp hatched larvae within 24 hours twice daily, water pH: 6-8.5, variation within 1.0, TOC: less than 2 mg / l, total hardness: calcium carbonate concentration 10-250 mg / l.

  In the present invention, the acceptable range means that the t-test of the gene expression level at the significance level of 5%, preferably at the significance level of 1%, more preferably at the significance level of 0.1%, results in the normal control and the test biological sample. Within a range where there is no significant difference between them. That is, the gene expression profile typically found in aquatic organisms with good physiological conditions. The control level can be a database of expression patterns based on aquatic organisms that have already been tested.

  In the present invention, aquatic organisms having a good physiological condition are grown under breeding conditions in accordance with the International Standard for Toxicity Test of the Chemical Substances Control Law, and there are no behaviorally or apparently abnormal individuals, and An aquatic organism whose overall mortality rate is lower than 5% for 7 consecutive days during acclimatization and whose mortality rate in the control group does not exceed 10% at the end of the toxicity test. In the present invention, “aquatic organisms having a favorable physiological state” can also be referred to as “aquatic organisms in a standard state”.

  As a third step of this method, whether or not the physiological condition of the test aquatic organism is good is evaluated based on the comparison result of the expression level. In the present invention, “evaluating whether or not the physiological state of the test aquatic organism is good” means the same meaning as “indicating whether or not the physiological state of the test aquatic organism is good”. .

  An increase or decrease in the expression level of the aquatic organism gene involved in the physiological state in the biological sample derived from the test aquatic organism is an indicator of whether or not the physiological state of the subject aquatic organism is good. For example, a decrease in the expression level of the gene comprising the base sequence set forth in any one of SEQ ID NOs: 1 to 93 in a biological sample derived from a test aquatic organism compared to a normal control level, or SEQ ID NOs: 93 to An increase in the expression level of the gene containing the base sequence described in any of 111 is an indicator that the physiological state of the test aquatic organism has deteriorated. On the contrary, the expression level of the gene containing the base sequence described in any one of SEQ ID NOs: 1-111 in the biological sample derived from the test varicella organism is equivalent to the normal control level. It is an indicator that the physiological state of the organism is good.

  If one or more of the aquatic organism genes involved in the physiological condition is compared to a normal control level and the expression level is altered in a biological sample from the subject aquatic organism, It is an indicator that the physiological condition is deteriorating. For example, at least 1%, 5%, 10%, 25%, 50%, 60%, 80% of Minamata organism genes (base sequences described in any of SEQ ID NOs: 1-111) involved in physiological conditions , 90%, or more can be assessed as deteriorating physiological status if they change from normal control levels.

  Specifically, when the expression level of the varicella organism gene group involved in the physiological state selected from the base sequences set forth in any of SEQ ID NOs: 1 to 93 is lower than the normal control expression level, the test varicella It can be evaluated that the physiological state of the organism is not good.

  More specifically, the expression level of a gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 1 to 23 is 50% (1 / If it is smaller than 2 times, more preferably less than 25% (1/4 times), it can be evaluated that the physiological state of the test aquatic organism is not good. Moreover, the expression level of the gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 1 to 93 is more than 50% (1/2 times) the normal control expression level. When it is small, it can be evaluated that the physiological state of the test aquatic organism is not good. Alternatively, using statistical methods, if a significant difference is observed in the decrease in gene expression level at a significance level of 5%, preferably a significance level of 1%, more preferably a significance level of 0.1%, It can be evaluated that the physiological state of the organism is not good.

  In addition, when the expression level of the aquatic organism gene involved in the physiological state selected from the base sequences set forth in any of SEQ ID NOs: 93 to 111 is higher than the normal control expression level, the physiological state of the test aquatic organism Can be evaluated as not good. More specifically, the expression level of a gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 93 to 111 is 200% (2 times the normal control expression level). ) Larger than that, it can be evaluated that the physiological state of the test aquatic organism is not good. Alternatively, if a significant difference in gene expression level is observed at a significance level of 5%, preferably a significance level of 1%, more preferably a significance level of 0.1%, using a statistical method, It can be evaluated that the physiological state of the organism is not good.

  In the present invention, a diagnostic agent for evaluating the physiological state of aquatic organisms is also provided. The diagnostic agent of the present invention includes a compound that binds to the polynucleotide or polypeptide of the present invention. Preferably, an oligonucleotide that hybridizes to the base sequence described in any one of SEQ ID NOs: 1 to 138 or an antibody that binds to a protein encoded by the base sequence described in any one of SEQ ID NOs: 1 to 138, It can be used as such a compound.

  Are the aquatic organism genes involved in the physiological state identified in the present specification suitable as an indicator organism in which the test aquatic organism is a standard indicator in a biological test or a control group in a biological test? It can also be used when evaluating whether or not.

The present invention includes the following steps (a) to (c), and the test aquatic organism is suitable as an indicator organism in a standard state in a biological test or an indicator organism to be a control group in a biological test. It relates to a method for evaluating whether or not.
(A) a step of determining an expression level of aquatic organism genes involved in a physiological state in a biological sample derived from a test aquatic organism (b) an expression level of the gene in a biological sample derived from a test aquatic organism According to the comparison result of the expression level in the steps (c) and (b) of comparing the normal control expression level of the gene, the test varicella organism is A process to evaluate whether it is suitable as an indicator organism to be a control

  As the first step of this method, the expression level of at least one of the aquatic organism genes involved in the physiological state is determined in a biological sample derived from the subject aquatic organism. In the present method, the gene defined above can be selected as the “aquatic organism gene involved in physiological state”, and “determination of gene expression level” can also be performed by the method defined above.

  As a second step of the method, the expression level of one or more aquatic organism genes involved in the physiological state in a biological sample from the test aquatic organism is compared to the normal control level of the gene.

  As a third step of this method, whether the test aquatic organism is suitable as an indicator organism in a standard state in a biological test or an indicator organism to be used as a control group in a biological test based on the comparison result of the expression level. Evaluate whether or not. In the present invention, “evaluating whether or not it is suitable as an indicator organism in a standard state in a biological test, or an indicator organism in a control group in a biological test” means “in a standard state in a biological test. It indicates the same meaning as “indicating whether or not it is suitable as an indicator organism or an indicator organism that serves as a control group during a biological test”.

  If one or more of the aquatic organism genes involved in the physiological condition is compared to a normal control level and the expression level is equivalent in a biological sample from the subject aquatic organism, the subject aquatic organism is It can be evaluated that it is suitable as an indicator organism in a standard state in a biological test or as an indicator organism to be used as a control group in a biological test. For example, at least 1%, 5%, 10%, 25%, 50%, 60%, 80% of Minamata organism genes (base sequences described in any of SEQ ID NOs: 1-111) involved in physiological conditions , 90%, or more, is equal to the normal control level, the test aquatic organism is a standard indicator organism in a biological test, or an indicator organism that serves as a control in a biological test. Can be evaluated as suitable.

  Specifically, when the expression level of the aquatic organism gene group involved in the physiological state selected from the base sequences set forth in any of SEQ ID NOs: 1 to 93 in the test aquatic organism is smaller than the normal control expression level Therefore, it can be evaluated that the test aquatic organism is not suitable as a standard indicator organism in a biological test or a reference indicator organism in a biological test.

  More specifically, the expression level of a gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 1 to 23 is 50% (1 / 2), more preferably less than 25% (1/4), the test aquatic organism is a standard indicator organism in a biological test, or a control group in a biological test. It can be evaluated that it is not suitable as an indicator organism. Moreover, the expression level of the gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 1 to 93 is more than 50% (1/2 times) the normal control expression level. If small, it can be evaluated that the test aquatic organism is not suitable as a standard indicator organism in a biological test or as a reference indicator organism in a biological test. Alternatively, using statistical methods, if a significant difference is observed in the decrease in gene expression level at a significance level of 5%, preferably a significance level of 1%, more preferably a significance level of 0.1%, It can be assessed that the organism is not suitable as a standard indicator organism in a biological test or as a control indicator organism in a biological test.

  In addition, when the expression level of the aquatic organism gene involved in the physiological state selected from the base sequence set forth in any of SEQ ID NOs: 93 to 111 in the test aquatic organism is higher than the normal control expression level, the test varicella It can be assessed that the organism is not suitable as a standard indicator organism in a biological test or as a control indicator organism in a biological test.

  More specifically, the expression level of a gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 93 to 111 is 200% (2 times the normal control expression level). ) Larger, it can be evaluated that the test aquatic organism is not suitable as a standard indicator organism in a biological test or as a reference indicator organism in a biological test. Alternatively, if a significant difference in gene expression level is observed at a significance level of 5%, preferably a significance level of 1%, more preferably a significance level of 0.1%, using a statistical method, It can be assessed that the organism is not suitable as a standard indicator organism in a biological test or as a control indicator organism in a biological test.

  The Minamata organism gene involved in the physiological state identified in the present specification is used to evaluate whether the test breeding conditions are suitable as conditions for breeding the indicator organism in the standard state in the biological test. Can also be used.

The present invention is a method for evaluating whether or not a test breeding condition includes the following steps (a) to (c) and is suitable as a condition for breeding a standard indicator organism in a biological test: About.
(A) a step of determining the expression level of aquatic organism genes involved in physiological conditions in a biological sample derived from aquatic organisms cultivated under test breeding conditions (b) biology derived from aquatic organisms to be examined The comparison of the expression level of the gene in the experimental sample with the normal control expression level of the gene (c) (b) shows that the test breeding condition is an indicator organism of the standard state in the biological test. To evaluate whether it is suitable as a condition for breeding

  As a first step of this method, in a biological sample derived from a test aquatic organism bred under a test breeding condition, the expression level of at least one of the aquatic organism genes involved in the physiological state is determined. . In the present method, the gene defined above can be selected as the “aquatic organism gene involved in physiological state”, and “determination of gene expression level” can also be performed by the method defined above.

  In this method, various conditions can be set as test breeding conditions. Conditions to be set include water temperature, dissolved oxygen concentration of water, light / dark cycle, type of feeding, water pH, total organic carbon (TOC), water hardness, and the like.

  As a second step of the method, the level of expression of one or more aquatic organism genes involved in the physiological state in a biological sample derived from a test aquatic organism bred under test breeding conditions is determined as a normal control of the gene. Compare with level.

  As a third step of the method, whether or not the test breeding condition is suitable as a condition for breeding the indicator organism in the standard state in the biological test is evaluated based on the comparison result of the expression level. In the present invention, “evaluating whether or not the test breeding conditions are suitable as conditions for breeding the indicator organism in the standard state in the biological test” means that “the test breeding conditions are the standard in the biological test. It indicates the same meaning as “indicates whether or not it is suitable as a condition for raising an indicator organism of a state”.

  When one or more of the aquatic organism genes involved in physiological conditions are compared to normal control levels and the expression levels are equivalent in biological samples derived from test aquatic organisms that have been bred under test breeding conditions Therefore, it can be evaluated that the test breeding conditions are suitable as conditions for breeding the indicator organism in the standard state. For example, in a biological sample derived from a test aquatic organism bred under test breeding conditions, among aquatic organism genes involved in physiological state (base sequence described in any one of SEQ ID NOs: 1-111), For breeding standard indicator organisms if at least 1%, 5%, 10%, 25%, 50%, 60%, 80%, 90%, or more are equivalent to normal control levels It can be evaluated that it is suitable as a condition of

  Specifically, the expression level of the aquatic organism gene group involved in the physiological state selected from the base sequences set forth in any one of SEQ ID NOs: 1 to 93 in test aquatic organisms bred under test breeding conditions is When the expression level is lower than the normal control level, it can be evaluated that the test breeding conditions are not suitable as conditions for breeding the indicator organism in the standard state in the biological test.

  More specifically, the expression level of a gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 1 to 23 is 50% (1 / If it is less than 2 times, more preferably less than 25% (1/4 times), the test breeding conditions are not suitable as conditions for breeding the standard indicator organism in the biological test. Can be evaluated. Moreover, the expression level of the gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 1 to 93 is more than 50% (1/2 times) the normal control expression level. When it is small, it can be evaluated that the test breeding condition is not suitable as the condition for breeding the standard indicator organism in the biological test. Alternatively, using statistical methods, if there is a significant difference in the decrease in gene expression level at a significance level of 5%, preferably a significance level of 1%, more preferably a significance level of 0.1%, It can be evaluated that the condition is not suitable as a condition for raising an indicator organism in a standard state in a biological test.

  In addition, the expression level of the aquatic organism gene involved in the physiological state selected from the base sequences set forth in any of SEQ ID NOs: 93 to 111 in the test aquatic organisms bred under the test breeding conditions is normal control expression When the level is higher than the level, it can be evaluated that the test breeding conditions are not suitable as conditions for breeding the standard indicator organism in the biological test.

  More specifically, the expression level of a gene group consisting of at least one or more base sequences selected from the base sequences set forth in any of SEQ ID NOs: 93 to 111 is 200% (2 times the normal control expression level). If it is greater than the normal control expression level, it can be assessed that the test breeding conditions are not suitable as conditions for breeding the standard indicator organism in the biological test. Alternatively, if statistical techniques are used and there is a significant difference in gene expression level at a significance level of 5%, preferably a significance level of 1%, more preferably a significance level of 0.1%, It can be evaluated that the condition is not suitable as a condition for raising an indicator organism in a standard state in a biological test.

  The present invention relates to a method for growing aquatic organisms under test breeding conditions evaluated as being suitable as conditions for breeding indicator organisms in a standard state in the above biological test. Aquatic organisms grown by this method can be used as standard state indicator organisms in various biological tests.

  The present invention relates to a nucleic acid microarray comprising one or a plurality of probes corresponding to the nucleic acid sequence described in any one of SEQ ID NOs: 1 to 138.

  A nucleic acid microarray is a convenient device for comparing the expression levels of a large number of genes simultaneously. Expression profiling based on nucleic acid microarrays can be performed by the method disclosed in, for example, “Microarray Biochip Technology” (Mark Schena, Eaton Publishing, 2000).

Nucleic acid microarrays include immobilized high density probes for detecting multiple genes. Thus, the expression levels of many genes can be estimated simultaneously by a single round of analysis. In the method of the present invention, gene expression may be determined by a nucleic acid microarray. The nucleic acid microarray-based method of the present invention includes the following steps:
(1) synthesizing aRNA or cDNA corresponding to the marker gene;
(2) a step of hybridizing aRNA or cDNA with a probe relating to a marker gene; and (3) a step of detecting aRNA or cDNA hybridized with the probe and quantifying the amount of mRNA.

  aRNA refers to RNA transcribed from a template cDNA by RNA polymerase. ARNA transcription kits for expression profiling based on nucleic acid microarrays are commercially available. With such a kit, aRNA can be synthesized from T7 promoter-binding cDNA as a template using T7 RNA polymerase. On the other hand, cDNA can be amplified by PCR using random primers using cDNA synthesized from mRNA as a template.

  The nucleic acid microarray of the present invention comprises a probe for detecting aquatic organism genes (marker genes) involved in the physiological state of the aquatic organism of the present invention. There is no limit on the number of probes for marker genes spotted on the nucleic acid microarray. For example, 1%, 5%, 10%, 25%, 50%, 60%, 80%, 90% or more of the nucleic acid sequence (gene) described in any one of SEQ ID NOs: 1-111 of the present invention The above genes (more specifically, 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 20, 23, 25 , 40, 50, 75, 100 or more genes) may be spotted. In addition to the marker gene, probes corresponding to other genes can be spotted on the DNA molecule chip. For example, a gene probe whose expression level hardly changes may be spotted on a nucleic acid microarray. If it is intended to compare assay results between multiple arrays or between different assays, such genes can be used to normalize the assay results.

  Probes are designed for each selected marker gene and spotted on a nucleic acid microarray. Such a probe may be an oligonucleotide comprising, for example, 5 to 50 nucleotide residues. Methods for synthesizing such oligonucleotides on nucleic acid microarrays are known to those skilled in the art. Longer DNA can be synthesized by PCR or chemically. A method for spotting long DNA synthesized by PCR or the like on a slide glass is also known to those skilled in the art. The nucleic acid microarray obtained by the above method can be used in the evaluation method of the present invention.

  After the prepared nucleic acid microarray is brought into contact with aRNA, hybridization between the probe and aRNA is detected. The aRNA can be pre-labeled with a fluorescent dye. ARNA can be labeled with fluorescent dyes such as Cy3 (red) and Cy5 (green). The aRNA from the test aquatic organism and the control are each labeled with a different fluorescent dye. The difference between both expression levels can be estimated based on the difference in signal intensity. A fluorescent dye signal on the nucleic acid microarray can be detected by a scanner and analyzed using a special program. For example, Affymetrix Suite can be listed as a software package for nucleic acid microarray analysis.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[Example 1] Confirmation of influence of feed on physiological state of medaka The effect of breeding with different diets on the physiological state of medaka was examined. Specifically, medaka was reared in an environment where the conditions other than the feed to be fed were the same, and the survival rate of medaka and the expression level of each gene of medaka were compared.
First, medaka was bred by the medaka growth method described below, and the survival rates when each feed was given were compared.

<Growing method of medaka>
For ecotoxicity studies, mature medaka from 2 to 6 months of age is usually used for research. Medaka standard strain (NIES strain) was obtained from National Institute for Environmental Studies (Japan), spawned at National Institute of Advanced Industrial Science and Technology (Japan), and the obtained F1 generation was used for studying the growth method of medaka. The results of preliminary experiments revealed that there was a large difference in gene expression between males and females (experimental results not shown), so males and females were examined separately. The aquarium used for breeding was a 45-60 cm aquarium made of glass or a chemically inert material (eg, made of fluororesin). During breeding, the water temperature was 24 ± 1 ° C., the photoperiod was 16 hours light and 8 hours dark, aeration was performed so that the dissolved oxygen concentration did not fall below 80%, and the feed was fed twice a day. As feed, "Artemia within 24 hours of hatching (Artemia) product name: brine shrimp (Nisshin Marine Tech, Japan)", "Product name: Otohime (Nisshin Marubeni Feed, Japan)", "Product name: Tetramine (German Tetra, Germany) "or" Product Name: Medaka's Food (Itosui, Japan) "was used.

  For breeding water, tap water for drinking was filtered through a PES membrane cartridge (TCS-E020, ADVANTEC) and an activated carbon cartridge filter (TCC-WL-SOCP, ADVANTEC), and then aerated for a day or more. These filters were changed at least once a month. The density of the fish in the breeding aquarium did not exceed 100 fish / 50L, and the breeding tank was changed once a week. The total hardness of the breeding water during the breeding period is preferably a calcium carbonate concentration of 10 to 250 mg / L and a pH in the range of 6 to 8.5, but the breeding water used so far has a total hardness of 50 to 70 mg / L L, pH 7-8.

A comparison of the survival rate when each feed is given is shown in FIG. As a result, it was clarified that, unlike Artemia, when Otohime, Tetramine, and Medaka feed were fed as feed, the survival rate was remarkably lowered and the growth state was also lowered. That is, it was clarified that the kind of feed to be given greatly affects the survival rate and growth state of medaka. In addition, it is clear from these results that it is optimal to give Artemia as a feed when cultivating medaka in a standard state (medaka with high survival rate and health condition) that is the subject of ecotoxicity tests. It became.
Next, the gene expression state of each medaka that was fed and bred with the above-described breeding method was confirmed by DNA microarray analysis. Microarray analysis was performed by the following steps.

<Nucleic acid extraction>
For extraction of medaka total RNA, RNeasy Lipid Tissue Midi Kit (QIAGEN) was used. The outline of the procedure is as follows. That is, a frozen medaka individual (about 3 cm long) was pulverized in a mortar while adding liquid nitrogen, and half of the powder was added to 5 mL of QIAzol Lysis Reagent, rapidly stirred vigorously, and left at room temperature for 5 minutes. 1 mL of chloroform was added and stirred for about 15 seconds, then placed at room temperature for 2 to 3 minutes, and the supernatant was separated by centrifugation. To this supernatant, 3 mL of 70% ethanol was added and mixed well, and the entire amount was passed through an RNeasy Midi spin column. The column was washed with 4 mL Buffer RW1 and 2.5 mL Buffer RPE, and finally RNA was eluted with 300 μL of RNase-free water.

<Labeling of medaka total RNA>
According to the method of Eberwine et al. (Eberwine, et al., PNAS 89: 3010 (1992)), biotin-labeled cRNA was obtained from total RNA extracted from medaka. That is, mRNA-derived cDNA was prepared by reverse transcription reaction from 10 ug of total RNA using T7-oligo (dT) as a primer. Thereafter, In Vitro Transcription reaction was performed with T7 RNA polymerase to prepare cRNA. By adding biotinylated cytosine and biotinylated uracil as substrates during the in vitro transcription reaction, biotin was incorporated into cRNA and labeled. cRNA was fragmented by 50 to 200 bases by hydrolysis.

<Synthesis of DNA microarray with medaka EST sequence>
The DNA microarray was produced by using a maskless photosynthetic DNA microarray production technology (hereinafter referred to as MAS) manufactured by NimbleGen Systems (USA). This technology is a microarray fabrication technology developed by Singh-Gasson et al. (Singh-Gasson, et al., Nat. Biotech. 17: 974 (1999)). UV light is emitted by Digital Light Processor (manufactured by Texas Instrument). By selectively controlling, oligonucleotides having different base sequences can be synthesized on a slide glass. By synthesizing hundreds of thousands of oligonucleotides with different sequences on a slide glass, a DNA microarray for comprehensively detecting gene expression can be prepared.

  This time, in order to create a DNA microarray based on Medaka's EST, the base sequence of EST, which is the basic information for the design, used the information registered in TIGR (The Institute for Genomic Research). As of May 17, 2004, there are 26,689 unique EST sequences registered in TIGR. After examining the specificity based on these EST base sequence information, seven types of base sequence portions specific to each EST were selected for one EST in units of 60 bases. The selected EST-specific nucleotide sequence was synthesized with MAS on a slide glass by MAS. Finally, 7 types of 60 base sequences per EST, a total of 186,823 oligonucleotides (hereinafter referred to as probes) were synthesized for 26,689 types of ESTs as a medaka EST sequence-equipped DNA microarray.

<Hybridization>
Hybridization of the probe on the microarray equipped with medaka EST sequence and fragmented biotin-labeled cRNA was performed under the following conditions. A 40% formamide solution containing 10 ug of fragmented biotin-labeled cRNA was brought into contact with the microarray and incubated at 42 ° C. for 14-16 hours. Thereafter, the slide glass was washed, and streptavidin-Cy3 was reacted at room temperature for 25 minutes, and Cy3 was further bound to the fragmented biotin-labeled cRNA bound to the probe on the microarray.

<Microarray scanning and data conversion>
The microarray after hybridization was subjected to a digitization process by measuring fluorescence intensity of Cy3 on the microarray with a DNA microarray scanner GenePix 4000B (manufactured by Axon Instruments). The gene expression intensity of each EST was the average value of the fluorescence intensity of 7 types of probes per EST.

  Using the above process, the expression intensity of several male and female male medaka bred on the recommended diet “Artemia” was examined using a DNA microarray. The expression intensity of two individuals (Artemia breeding male 1, Artemia breeding male 2) was compared using plot diagrams. As a result, it was clarified that Artemia breeding male 1 and Artemia breeding male 2 show the same level of expression intensity in most genes (FIG. 1a). In addition, using the same technique, we examined the expression intensity of several male and female medaka males bred with "Medaka feed", a feed different from Artemia, using a DNA microarray. The expression intensity of two individuals (medaka feed-fed male 1 and medaka feed-fed male 2) was compared using plot diagrams. As a result, it was revealed that medaka food-fed male 1 and medaka food-fed male 2 showed the same level of expression in most genes (FIG. 1b). That is, when the same kind of feed was given in the same environment, it became clear that many genes showed the same level of expression.

  Next, we compared medaka-feeding osmedaka and artemia-grown osmedaka. The expression strength of two individuals (medaka feed-fed male 1 and artemia-fed male 1) was compared using a plot diagram. It became clear that the gene expression was different (Fig. 1c). Furthermore, it was clarified that the medaka bred with different feeds (Otohime and Tetramine) showed a different gene expression situation than when bred with the recommended feed Artemia.

  Based on the above results, the gene expression status of mature medaka varies greatly depending on the feed even when acclimatized in the same environment, and the expression status of each gene correlates with the survival rate and growth status of medaka Became clear.

Example 2 Confirmation of Effect of Low Dissolved Oxygen Concentration on Physiological State of Medaka Next, the influence of low dissolved oxygen treatment on the physiological state of medaka was examined. Specifically, medaka were reared in an environment where the conditions other than the dissolved oxygen concentration were the same, and the survival rate of medaka and the expression level of each gene of medaka were compared.

Breeding in the same environment as that of the control group during the ecotoxicity test (breeding under saturated dissolved oxygen concentration) or breeding under low dissolved oxygen concentration was performed as follows.
The same environment (control group) as that in the control group during the ecotoxicity test was carried out by a semi-water-stop type (daily change of water every day). 2 L of test water was added to a 3 L glass beaker, and 7 fish were raised per beaker. The medaka used was acclimatized with Artemia, and feeding was stopped 24 hours before the test, and no feed was given during the test period. Aeration was performed during the test period, and water was changed once a day. In this state, it was confirmed that the saturated dissolved oxygen concentration was observed throughout the 96 hour test.
After 96 hours of testing, 5 males and 5 females were frozen in liquid nitrogen and stored as they were.

When performing low-dissolved oxygen treatment, the test water was sealed and oxygen was consumed by breathing medaka. When the dissolved oxygen concentration reached 10%, oxygen was supplied to maintain the dissolved oxygen concentration at 10% for 96 hours. Under this condition, 8 of the 9 males and 9 females out of a total of 18 died. Surviving individuals were frozen in liquid nitrogen and stored as such.
From the above results, it was clarified that the dissolved oxygen concentration greatly affects the survival rate of medaka and is one of the important factors for maintaining the medaka's health.

  Next, the gene expression state of each medaka bred under different dissolved oxygen concentrations was confirmed by DNA microarray analysis using the above breeding method. Extraction of nucleic acid from the stored sample, labeling of medaka total RNA, synthesis of DNA microarray with medaka EST sequence, hybridization, scanning of microarray and data conversion were carried out by the steps described in Example 1.

  The expression intensity of 26,689 genes was examined using a DNA microarray in mature male medaka (control group) males and females reared in the same environment as the control group under ecological toxicity tests. When the expression intensity of two individuals (control male 1 and control male 2) was compared using a plot, the control male 1 and control male 2 had almost the same expression intensity. Similarly, the expression intensity of several male and female medaka bred under low dissolved oxygen concentration was examined using a DNA microarray. Comparing the expression intensities of two individuals (low dissolved oxygen concentration treatment male 1, low dissolved oxygen concentration treatment 2), low dissolved oxygen concentration treatment male 1 and low dissolved oxygen concentration treatment male 2 have almost the same expression intensity. It became clear that there was. That is, it was revealed that many genes show the same level of expression when reared under the same dissolved oxygen concentration in the same environment.

  In addition, the low-dissolved oxygen concentration-treated osmedaka and the control osmedaka were compared. As a result of comparing the expression intensity of two individuals (low dissolved oxygen concentration-treated male 1, control group male 1) using a plot diagram, the expression of various genes in low dissolved oxygen concentration treated male 1 and control group male 1 It became clear that the situation was different (Fig. 1d).

  From the above results, the gene expression status of mature medaka varies greatly depending on the dissolved oxygen concentration even when acclimated in the same environment, and the expression status of each gene correlates with the survival rate and growth status of medaka It became clear.

[Example 3] Identification of genes that serve as indicators of medaka health status (survival rate and / or breeding status) In Example 1 and Example 2, the expression status of each gene of medaka depends on the survival rate and growth status of medaka It became clear that there was a correlation. When the survival rate and growth state of medaka were deteriorated, genes whose expression levels were greatly changed were identified.
When comparing the expression level of a specific gene between two different breeding environments, the average value of the fluorescence intensity is calculated from the expression level of the gene in each breeding environment from seven types of probes per gene. Differences were confirmed by t-test. From the t-test, it was determined that the gene expression was changed between the two breeding environments for those having a significance probability of 0.1% or less (significance level 0.1%). From this gene group with a significance probability of 0.1% or less, genes whose actual fluorescence intensity values were changed to 1/4 times or less, or 1/2 times or less were identified as genes whose expression level was greatly changed. .

  From 26,689 types of genes, 264 types of genes were significantly different at a significance level of 0.1% without being influenced by factors such as the difference in dissolved oxygen concentration and food. Of these 264 genes, the genes whose fluorescence intensity changed to ¼ times or less changed to 23 ½ times or less, regardless of factors such as the difference in dissolved oxygen concentration and food. There were 93 kinds of genes and 18 kinds of genes that had changed more than twice.

In addition, as a negative control, a gene was identified in which the expression level of the gene does not change even in an environment that is thought to affect the physiological state of medaka. The identification method of these genes is as follows. The difference in the expression level of the Minamata organism gene involved in the physiological state and the normal control expression level is within ± 20% (80 to 120%) of the normal control expression level in all test conditions, and the t-test A gene having a significant probability of 5% or more (fluorescence intensity: 2 ⁶ to 2 ¹¹ = 64 to 2048) was identified. Further, the difference between the expression level of the Minamata organism gene involved in the physiological state and the normal control expression level is within ± 30% (70 to 130%) of the normal control expression level in all test conditions, and the significance probability is 1% or more of genes (fluorescence intensity: 2 ¹⁰ to = 1024) were identified. Twenty-seven genes identified by these methods were identified as genes whose expression level was not affected by the physiological state of medaka.

  Genes whose expression levels were changed to ¼ times or less are shown in SEQ ID NOs: 1 to 23 in Table 1, and genes whose expression levels were changed to ½ times or less are shown in SEQ ID NOs: 1 to 93 in Table 1. The genes that have changed more than twice are shown in SEQ ID NOs: 93 to 111 in Table 1. The gene as a negative control is shown in Table 2 (SEQ ID NOs: 112 to 138).

[Example 4] Confirmation of influence of pH (hydrogen ion concentration) on physiological state of medaka The influence of the pH of breeding water on the physiological state of medaka was examined. Specifically, medaka was bred in an environment with the same conditions other than the pH of the breeding water (acidic conditions and alkaline conditions), and changes in the expression of aquatic organism genes involved in physiological conditions were confirmed.

Rearing under acidic conditions and alkaline conditions was performed as follows.
For the test, males and females of about 5 months after hatching, which were transferred from the National Institute for Environmental Studies and were subcultured, were used. After appropriately adjusting the pH of 5 L of activated carbon filtered water, nine male and female females were added and tested for 96 hours. Using a pH controller (Biott DT-1023), 0.1N hydrochloric acid was appropriately added for acidic conditions and 0.1M sodium hydroxide was appropriately added for alkaline conditions so that the pH would be constant. HORIBA Navi D-55 was used in combination with pH monitoring. Water exchange was performed at a rate of about 100-150 ml / h to dilute the salt concentration during the test period. The aeration was performed, the water temperature was 24 ± 1 ° C., the light cycle of 16 hours light and 8 hours dark was maintained, and feeding was stopped after 24 hours from the start of the test.

As a result of the preliminary test, the test was carried out under the condition that pH 4.0, which is the condition where about half of the fish die, was set as an acidic condition and pH 10.8 was set as an alkaline condition. The number of dead fish after 96 hours at pH 4.0 is 4/9 fish (male) and 6/9 fish (female), and at pH 10.8, 5/9 fish (male) and 5/9 fish ( Female). Samples were frozen in liquid nitrogen, and all individuals were pulverized to extract RNA and used for experiments.
From the above results, it was clarified that the pH of the breeding water has a great influence on the survival rate of medaka and is one of the important factors for maintaining the health of medaka.

  Next, the gene expression state of each medaka bred under different pH conditions was confirmed by DNA microarray analysis. Extraction of nucleic acid from the stored sample, labeling of medaka total RNA, synthesis of DNA microarray with medaka EST sequence, hybridization, scanning of microarray and data conversion were carried out by the steps described in Example 1.

  Based on the results of these DNA microarray analyses, the expression status of Minamata organism genes (SEQ ID NOs: 1-111) involved in the physiological state identified in Example 3 was confirmed (Table 3). The expression status of these genes agreed well with the pattern of gene expression (Table 1) in which the physiological state was not good. That is, it was proved that the aquatic organism gene involved in the physiological state identified in the present invention is more useful and reliable.

[Example 5] Confirmation of the effect of high dissolved oxygen on the physiological state of medaka The concentration of dissolved oxygen in breeding water affects the physiological state of medaka in both low and high dissolved oxygen conditions Have been reported (Lushchak VI, et al., Comp Biochem Physiol B Biochem Mol Biol. 144: 283 (2006), Epub 2006 Jun 5). It has also been reported that gene expression changes due to oxidative stress under highly dissolved oxygen conditions (Olsvik PA, et al., J Exp Biol. 209: 2893 (2006)). Therefore, in addition to the confirmation of the effect of low dissolved oxygen shown in Example 2, the effect of high dissolved oxygen on the physiological state of medaka was confirmed. Specifically, medaka were reared in an environment where conditions other than the dissolved oxygen concentration were the same, and changes in the expression of aquatic organism genes involved in physiological conditions were compared.

Breeding under high dissolved oxygen conditions was performed as follows.
For the test, males and females about 4 months old after hatching, which were transferred from the National Institute for Environmental Studies and were subcultured, were used. Bubbling of 5 L of activated carbon filtered water from an oxygen cylinder was performed overnight, and then 9 males and 10 females were introduced for a 96-hour test. During the test period, bubbling from the oxygen cylinder was continued, and activated carbon filtered water in which bubbling from the oxygen cylinder was performed overnight also at the time of water change performed once every day was used. Furthermore, the test was conducted by maintaining the water temperature of 24 ± 1 ° C., the light cycle of 16 hours light and 8 hours dark, and stopping feeding after 24 hours from the start of the test.

  As a result of the preliminary test, the dissolved oxygen concentration of activated carbon filtered water bubbling the atmospheric air overnight was 8.32 mg / L, and the dissolved oxygen concentration of activated carbon filtered water bubbling oxygen from the oxygen cylinder overnight was 40.29 mg / L. . METTLER TOLEDO SG6 was used to measure these dissolved oxygen concentrations.

  As a result of the 96-hour high-dissolved oxygen test, none of the animals showed death or behavioral abnormalities. From this result, it was found that the high dissolved oxygen condition did not affect the survival rate. Surviving individuals were frozen in liquid nitrogen for 5 males and 5 females each, and all individuals were pulverized, and RNA was extracted and used for experiments.

  Next, the gene expression state of each medaka bred under different dissolved oxygen conditions was confirmed by DNA microarray analysis. Extraction of nucleic acid from the stored sample, labeling of medaka total RNA, synthesis of DNA microarray with medaka EST sequence, hybridization, scanning of microarray and data conversion were carried out by the steps described in Example 1.

  Based on the results of these DNA microarray analyses, the expression status of Minamata organism genes (SEQ ID NOs: 1-111) involved in the physiological state identified in Example 3 was confirmed (Table 3). The expression status of these genes agreed well with the pattern of gene expression (Table 1) in which the physiological state was not good. From this, it was shown that breeding under high dissolved oxygen conditions deteriorates the physiological state of medaka. Furthermore, it was shown that the physiological state of the medaka can be evaluated regardless of the survival rate by using the aquatic organism gene related to the physiological state identified in the present invention.

“▽ (black triangle)” in Table 1 indicates that the expression level of the gene in the test aquatic organism decreased compared to the normal control group, and “△” indicates the comparison with the normal control group. This shows that the expression level of the gene in the test aquatic organism increased. In addition, the values from A to H on the horizontal axis in Table 1 indicate the ratios of the tendency intensities shown below. These values indicate the percentage (%) of how much the expression of the gene has changed when the gene expression in the normal control group is taken as 100%.
A: (Medakaos) Breeding in the same environment as the control group under ecotoxicity test / breeding with low dissolved oxygen concentration B: (Medakames) Breeding in the same environment as the control group during ecotoxicity test / low dissolved oxygen Breeding in Concentration C: (medakaos) reared in Artemia / bred in Otohime D: (medakames) reared in Artemia / bred in Otohime E: (medakaos) reared in Artemia / bred in tetramine F: (medakames) Artemia Breeding in / Tetramine Breeding G: (Medakaos) Breeding in Artemia / Breeding in Medaka Feeding H: (Medakames) Breeding in Artemia / Breeding in Medaka Feeding

The genes were narrowed down at a significance level of 0.1% (p <0.001, and).
“-” In Table 2 indicates that the expression level of the gene was not affected by the physiological state of medaka and was constant within a certain range. The “expression level” on the horizontal axis represents the average expression intensity of the gene under each test condition.

Table 3 shows how much the expression of the gene changed when the gene expression in the normal control group was taken as 100%. In the table, “▽ (black lower triangle)” and “Δ” have the following meanings and visually indicate numerical values.
“▽▽▽”: Gene expression level decreased to less than 25% compared to normal control group “▽▽”: Gene expression level decreased from 25% to less than 50% compared to normal control group “▽”: Gene expression level decreased by 50% or more and less than 100% compared to normal control group “ΔΔΔ”: Gene expression level increased by 400% or more compared to normal control group “ΔΔ”: Normal control group Compared to 200% or more and less than 400%, the gene expression level increases “Δ”: Gene expression level increases from 100% or more to less than 200% compared to the normal control group

It is a figure which shows the comparison of the gene expression condition of the medaka at the time of raising with Artemia. It is the figure which plotted the expression intensity of Medaka chaos 1 at the time of breeding by Artemia on the horizontal axis, and the expression intensity of Medaka chaos 2 at the time of breeding by Artemia on the vertical axis. When the gene expression intensities match, the gene plots are located on a straight line y = x with a slope of 45 degrees, indicating that the gene expression is well matched between the two tails. It is a figure which shows the comparison of the gene expression condition of the medaka at the time of raising with the food of a medaka. It is the figure which plotted the expression intensity of the medaka chaos 1 at the time of breeding with the feed of medaka on the horizontal axis, and the expression intensity of the medaka chaos 2 at the time of breeding with the feed of medaka on the vertical axis. It was revealed that the gene expression was well matched between the two fish. It is a figure which shows the comparison of the gene expression condition of the medaka when breeding with Artemia, and the gene expression situation of the medaka when breeding with food of medaka. It is the figure which plotted the expression intensity of the medaka chaos 1 at the time of breeding with the feed of Medaka with the horizontal axis | shaft and the expression intensity of the medaka chaos 1 at the time of breeding with the feed of medaka, plotted for every gene. It became clear that the expression of many genes differed between two fish. It is a figure which shows the comparison of the gene expression state of the medaka when rearing in the same environment as the control plot of an ecotoxicity test, and the gene expression state of the medaka when rearing under low dissolved oxygen. A plot of medachaos 1 expression intensity when bred in the same environment as the control zone of the ecotoxicity test on the horizontal axis and medachaos 1 expression intensity when treated with low dissolved oxygen on the vertical axis. is there. It became clear that the expression of many genes differed between two fish. After hatching, medaka was bred with four different diets (Artemia, Otohime, Tetramine, Medaka feed), and the effects on (A) survival rate and (B) growth state (body length) were examined. . When medaka was hatched and reared in Artemia, the survival rate was not decreased, the growth was good, and the medaka was considered to be in the standard state. On the other hand, when otome, tetramine, and medaka were fed, the survival rate was observed to be intermittently lowered although the degree was different.

Claims (16)

A method for evaluating the physiological state of a test aquatic organism, comprising the following steps (a) to (c).
(A) a step of determining an expression level of aquatic organism genes involved in a physiological state in a biological sample derived from a test aquatic organism (b) an expression level of the gene in a biological sample derived from a test aquatic organism The step of comparing the normal control expression level of the gene (c) and the step of evaluating whether or not the physiological condition of the test aquatic organism is good based on the comparison result of the expression level in (b) An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 23, and the expression level of the gene group in a test aquatic organism The method according to claim 1, wherein the physiological condition of the test aquatic organism is evaluated to be poor when the value of the expression level is lower than the normal control expression level. An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 93, and the expression level of the gene group in a test aquatic organism The method according to claim 1, wherein the physiological condition of the test aquatic organism is evaluated to be poor when the value of the expression level is lower than the normal control expression level. An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 93 to 111, and the expression level of the gene group in a test aquatic organism The method according to claim 1, wherein the physiological condition of the test aquatic organism is evaluated to be poor when the value of the expression level rises above the normal control expression level. Whether the test aquatic organism including the following steps (a) to (c) is suitable as an indicator organism in a standard state in a biological test or a reference organism in a biological test How to evaluate.
(A) a step of determining an expression level of aquatic organism genes involved in a physiological state in a biological sample derived from a test aquatic organism (b) an expression level of the gene in a biological sample derived from a test aquatic organism According to the comparison result of the expression level in the steps (c) and (b) of comparing the normal control expression level of the gene, the test varicella organism is A process to evaluate whether it is suitable as an indicator organism to be a control An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 23, and the expression level of the gene group in a test aquatic organism Is evaluated as being unsuitable as a standard indicator organism in a biological test or as a reference indicator organism in a biological test when the expression level falls below the normal control expression level. Method. An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 93, and the expression level of the gene group in a test aquatic organism Is evaluated as being unsuitable as a standard indicator organism in a biological test or as a reference indicator organism in a biological test when the expression level falls below the normal control expression level. Method. An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 93 to 111, and the expression level of the gene group in a test aquatic organism Is evaluated as not suitable as a standard indicator organism in a biological test or as a control indicator organism in a biological test when Method. A method for evaluating whether or not a test breeding condition including the following steps (a) to (c) is suitable as a condition for breeding an indicator organism in a standard state in a biological test.
(A) a step of determining the expression level of aquatic organism genes involved in physiological conditions in a biological sample derived from aquatic organisms cultivated under test breeding conditions (b) biology derived from aquatic organisms to be examined The comparison of the expression level of the gene in the experimental sample with the normal control expression level of the gene (c) (b) shows that the test breeding condition is an indicator organism of the standard state in the biological test. To evaluate whether it is suitable as a condition for breeding An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 23, and the expression level of the gene group in a test aquatic organism The method according to claim 9, wherein the test rearing condition is evaluated as unsuitable as a condition for rearing the indicator organism in the standard state in the biological test when the expression level falls below the normal control expression level. An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 1 to 93, and the expression level of the gene group in a test aquatic organism The method according to claim 9, wherein the test rearing condition is evaluated as unsuitable as a condition for rearing the indicator organism in the standard state in the biological test when the expression level falls below the normal control expression level. An aquatic organism gene involved in a physiological state is a gene group comprising at least one base sequence selected from the base sequences set forth in SEQ ID NOs: 93 to 111, and the expression level of the gene group in a test aquatic organism The method according to claim 9, wherein the test rearing condition is evaluated as unsuitable as a condition for rearing the indicator organism in the standard state in the biological test when the expression level is higher than the normal control expression level. A method for cultivating aquatic organisms under test breeding conditions evaluated as suitable for breeding indicator organisms in a standard state in a biological test by the method according to any one of claims 9 to 12. . The aquatic organism grown according to claim 13, which can be used as an indicator organism in a standard state in a biological test. The test varicella according to any one of claims 1 to 12, wherein a gene group consisting of at least one or more base sequences selected from the base sequences set forth in SEQ ID NOs: 112 to 138 is used as a negative control. A method for evaluating a physiological state of an organism, a physiological state of an indicator organism, or a breeding condition of a test aquatic organism / indicator organism. A nucleic acid microarray comprising one or more probes corresponding to the nucleic acid sequence of any one of SEQ ID NOs: 1 to 138.

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