KR101344810B1 - Biomarkers for evaluating sediment ecotoxicity of fullerene from aquatic midge, Chironomus riparius and the uses thereof - Google Patents

Abstract

The present invention is one or more funnels selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 72 ( Chironomus riparius ) biomarkers for fullerene including genes derived from genes, gene chips for fullerene ecological toxicity assessment, kits for fullerene ecological toxicity assessment, and compositions for fullerene ecological toxicity assessment and fullerene contamination Exposing the funnel to a sample to separate the RNA therefrom; Synthesizing cDNA using the separated RNA as a template; Contacting the synthesized cDNA with the gene chip to hybridize with the cDNA; Detecting a degree of hybridization between the gene chip and cDNA; And it provides a method for detecting the presence of fullerene in the sample comprising the step of comparing the detected degree of hybridization with the positive control group.

Description

Biomarkers for Evaluating sediment ecotoxicity of fullerene from aquatic midge, Chironomus riparius and the uses approx}

The present invention relates to invertebrate aquatic for carbon nanomaterial of ecotoxicity evaluation of the fullerene animal kkaldagu biomarkers and the use thereof, and more particularly, one or more kkaldagu is selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 to 72 (Chironomus riparius ) biomarker for the evaluation of fullerene (fullerene, C60) derived gene, gene chip for fullerene ecological toxicity evaluation, kit for fullerene ecological toxicity evaluation and composition for fullerene ecological toxicity evaluation and the biomarker It provides a method for detecting the presence of fullerene in the sample.

Nanotechnology is actively used in all fields of science as the development of science and technology allows the production of atomic-molecule-sized materials. However, despite the many benefits of nanotechnology, concerns about the harmfulness of nanomaterials have arisen internationally. Toxicity studies on nanomaterials are currently at the starting point, but mainly focused on human toxicity, and since the test method of nanotoxicity studies on soil organisms has not been properly developed, studies on soil organisms have been limited. . This is because nanomaterials are particulate matters unlike conventional water-soluble materials, which makes it difficult to apply standard toxicity test methods. In particular, fullerene, a representative carbon-based nanoparticle, refers to carbon cluster C60 formed by combining 60 carbon elements in the shape of a soccer ball. However, it is used as a resin to increase durability, heat resistance, remove static electricity, and noise filter. Recently, studies on the photoinduced toxicity (Yang et al., Toxicology 16 pp. 41 ~ 46 (2002)) and the possibility of inducing toxicity on aquatic ecosystem have been conducted. (Oberdorster, Environ Health Perspect 112 pp. 1058-1062 (2004)).

Chironomus is one of the most representative insect species in the aquatic ecosystem. The larvae of Suseonggi, which last up to 4th instar, are about 2 ~ 100 ㎜ in size and are an important indicator species of water pollution. They are used as food for fish and predatory invertebrates and birds and occupy an important position in the Suseo ecosystem food chain have. They are benthic organisms living mainly in junitos (sediments) and are easily exposed to accumulated pollutants such as PAHs (polycyclic aromatic hydrocarbons) in contact with pore water or upper layer water in sediments or inhaling sediments And is widely used for sediment toxicity testing. In particular, the introduction of nanomaterials, such as fullerene, a carbon nanoparticle that is representative of aquatic ecosystems, leads to the accumulation of sediment layers and toxic effects on benthic organisms that move between sediment layers and water. This will have an impact on the predators predominantly feeding benthic organisms, leading to the destruction of food chains in the ecosystem, resulting in a reduction in habitats, densities, species diversity, food, and growth of aquatic life. The larvae commonly used in this sediment toxicity test are the larvae of Chironomus riparius and Chironomous tentans , which survive for 10 days, grow rate or 28-day allegory to assess the effects of pesticide treatment. It is widely used for evaluating the number and sex ratio of an adult (Byung-Seok Kim et al., Korean Journal of Pesticide Science, Vol.

Funnel ( Chironomus riparius ) has long been used as an ecotoxicity monitoring model, but there is a limit to sensitive toxicity diagnosis because genetic information is not available. If functional chips are produced by the toxic material mechanism in the funnel, it is expected to make a significant contribution to the ecotoxicity and risk assessment. In addition, the development of a functional chip for fullerene, a carbon nanoparticle, is expected to be applicable to the toxicity of other types of carbon-based nanoparticles.

Korean Patent No. 1088456 discloses a method and kit for assessing environmental risks using an organism's ecotoxicity determination point, and Korean Patent No. 1183666 discloses a 'Songsari Genetic Chip for Environmental Hormone Toxicity'. However, as described in the present invention, no biomarker for ecotoxicological evaluation of fullerene, which is a nanoparticle of carbon material derived from a funnel, has not been found at all.

The present invention has been made in accordance with the above requirements, and the present inventors found that the expression level of specific genes in the fungus larvae belonging to the ecotoxicity indicator organisms exposed to fullerene 1 mg / L were significantly increased or decreased compared to the control without fullerene treatment. By confirming, the present invention was completed.

In order to solve the above problems, the present invention is one or more funnel selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 72 ( Chironomus The present invention provides a biomarker for ecotoxicity assessment of fullerene containing genes derived from riparius ), a gene chip for ecotoxicity assessment of fullerenes, and a kit for ecotoxicity assessment of fullerenes.

The present invention also provides a method for detecting the presence of fullerene in a sample using the biomarker.

In addition, the present invention provides a composition for evaluating the ecotoxicity of fullerenes comprising at least one genera derived gene selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 72.

The present invention provides a biomarker for evaluating ecotoxicity of a fullerene obtained from a fungus, an indicator species for which the sequence information is not yet known, and can be usefully applied to accurately determine ecotoxicity pollution caused by fullerene. It can greatly contribute to making improvement plans.

Figure 1 shows the SOLEXA sequencing method used to secure a specific expression genomic database of the funnel following fullerene treatment.
FIG. 2 shows genotyping of litter cells increased or decreased with fullerene treatment.

In order to achieve the above object, the present invention is a funnel ( Chironomus riparius ) provides a biomarker for evaluating the ecotoxicity of fullerenes (including fullerene).

Derived from the above The gene may be composed of one or more nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 72, but is not limited thereto. In addition, variants of the respective base sequences are included within the scope of the present invention. Specifically, the gene is at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 72, respectively. May comprise base sequences having at least 95% sequence homology. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

Each gene expression amount consisting of the nucleotide sequence of SEQ ID NOS: 1 to 72 that can be used as the biomarker of the present invention is characterized by a significant increase or decrease compared to the control group not treated with fullerene in vivo of the fungus exposed to fullerene Indicates.

The present invention also provides a gene chip for evaluating the ecotoxicity of fullerenes including the gene for the funnel.

Derived from the above The gene may consist of one or more nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 72, and preferably include all of the nucleotide sequences of SEQ ID NOs: 1 to 72, but is not limited thereto. In addition, variants of the respective base sequences are included within the scope of the present invention, as described above.

Gene chips can be used interchangeably with microarrays. In the microarray of the present invention, "microarray" refers to a microarray in which genes are immobilized at a high density on a substrate, and the genes are immobilized in a predetermined region, respectively. Such microarrays are well known in the art. Microarrays are described, for example, in U.S. Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The term "substrate" refers to any substrate to which a gene probe can be coupled under conditions that retain hybridization properties and keep the background level of hybridization low. Typically, the substrate may be a microtiter plate, a film (e.g., nylon or nitrocellulose) or a microsphere (bead) or chip. Before application or fixation to the membrane, the nucleic acid probe may be modified to promote immobilization or improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH ₂ groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

In addition, the present invention provides a kit for evaluating ecotoxicity of fullerenes comprising a gene of rugs.

Derived from the above The gene may consist of one or more nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 72, and preferably include all of the nucleotide sequences of SEQ ID NOs: 1 to 72, but is not limited thereto. In addition, variants of the respective base sequences are included within the scope of the present invention, as described above. The kit may include a reagent that separates RNA from a sample, a reagent that synthesizes cDNA from the separated RNA, and a hybridization reagent.

In addition, the present invention comprises the steps of exposing the funnel to the sample presumed to be contaminated with fullerene and separating RNA from it;

Synthesizing cDNA using the separated RNA as a template;

Contacting the synthesized cDNA with the gene chip to hybridize with the cDNA;

Detecting a degree of hybridization between the gene chip and cDNA; And

It provides a method for detecting the presence of fullerene in a sample comprising the step of comparing the detected degree of hybridization with a positive control.

The method of the present invention is a method for detecting the presence of fullerene in a sample presumed to be contaminated with fullerene, and the positive control group shows the nucleotide sequence of SEQ ID NOs: 1 to 72 of the present invention showing the increase or decrease of gene expression in the funnel exposed to fullerene. Refers to the degree of hybridization. The more similar the hybridization of the positive control group to the hybridization of the test sample, the higher the probability of fullerene in the sample. On the contrary, the less the similarity, the lower the probability of fullerene in the sample.

The funnel of the present invention preferably utilizes a funnel larvae, but is not limited thereto.

In the method for detecting the presence or absence of fullerene of the present invention, a method for separating RNA from a funnel exposed to a sample suspected to be contaminated with fullerene may use a method well known in the art.

As a method for synthesizing the first strand cDNA using the separated RNA as a template, a method commonly used in the art can be used. For example, using a reverse transcriptase, an RNase block ribonuclease inhibitor or the like, cDNA can be synthesized. Examples of reverse transcriptases include reverse transcriptases from various sources, such as avian myeloblastosis virus-derived virus reverse transcriptase (AMV RTase), mouse leukemia virus-derived reverse transcriptase ( murine leukemia virus-derived virus reverse transcriptase (MMLV RTase) and Rous-associated virus 2 reverse transcriptase (RAV-2 RTase).

Preferably, the cDNA can be labeled with a detectable labeling substance. The labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy5 or Cy3. When the first strand cDNA is synthesized by labeling Cy5 or Cy3 at the 5'-end of the primer, the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the reaction solution during the synthesis of the first strand cDNA, the synthetic product is synthesized and radioactive is incorporated into the synthesized product so that the synthesized product can be labeled with radioactivity have.

In addition, the present invention provides a composition for evaluating the ecotoxicity of fullerenes containing the gene of the funnel.

Derived from the above The gene may consist of one or more nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 72, and preferably include all of the nucleotide sequences of SEQ ID NOs: 1 to 72, but is not limited thereto. In addition, variants of the respective base sequences are included within the scope of the present invention, as described above.

The composition comprises one or more genera derived from the group selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 72 as an active ingredient, each of the genes in a control group that is not treated with fullerene in vivo in the incubation exposed to fullerene Compared with the fullerenes, it is possible to evaluate the toxicity of fullerenes.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One. Crumple  ( Chironomus riparius Pyro Sequencing to Obtain a Genome Database pyrosequencing )

50 larvae of caterpillars were exposed for 24 hours, lyophilized to extract mRNA, synthesized with cDNA, and used as a representative method of NGS (Next Generation Sequencing) using Pyro Sequencing (Roche 454, GS FLX Titanium Run). Secured the database.

① Preparation of Sample

Four samples were used to extract total RNA using Trizol. 1 ml of TRIzol Reagent was added to 50-100 mg tissue and completely homogenized. After phase separation, total RNA was precipitated, washed, and pellets were washed with RNase-free water. RNase-free water was added so that the final volume of 1 mg of total RNA was 0.5 ml, and then placed in a 65 ° C. heating block for 10 minutes. 3 μl of biotinylated-oligo (dT) probe and 13 μl of 20X SSC were added, then mixed well and cooled slowly at room temperature. Flick the SA-PMPs (Streptavidin-Paramagnetic Particles) tubes to flatten them well, then place the tubes on a magnetic stand to collect the SA-PMPs on the sides of the tubes, then carefully Was removed. In the same manner, SA-PMPs were washed three times using 0.5 × SSC (300 μl). Finally, SA-PMPs were resuspended in 100 μl of 0.5 × SSC. The prepared annealing reaction mixtures were all added to the washed tubes of SA-PMPs and reacted at room temperature for 10 minutes. At this time, inverting (inverting) and mixed every 1-2 minutes. The SA-PMPs tube was placed on a magnetic stand and the supernatant was carefully removed. In the same manner, SA-PMPs were washed four times using 0.1 × SSC (300 μl). Finally, the SA-PMP pellets were rediluted in 100 μl of RNase-free water, and the SA-PMPs tube was placed on a magnetic stand and the supernatant (eluted mRNA) was carefully transferred to a new tube. This process was repeated to obtain 200 μl of total eluted mRNA volume. 0.1 volume of 3M sodium acetate (pH 5.2) and 1.0 volume of isopropanol of the eluted mRNA amount were added and left overnight at −20 ° C., followed by centrifugation at 13,000 rpm and 4 ° C. for 10 minutes. mRNA pellets were washed with 1 ml of 75% ethanol (13,000 rpm, 5 min at 4 ° C.).

② cDNA synthesis

5 μl 10 × first strand buffer, 5 μl 100 mM DTT, 5 μl dNTP (2.5 mM each), 5 μl Oligo d (T) 20 (50 μM), 2.5 μl StrataScript Reverse Transcriptase ( 200 U / μl), xμl (<10μl) poly (A) + RNA (˜5 μg) were added to a final volume of 50 μl. After mixing well, the mixture was centrifuged, reacted at 65 ° C. for 3 minutes, and cooled at room temperature. The reaction was carried out at 42 ° C. for one hour. Sterile water was added to the inactivated first strand cDNA at 70 ° C. for 15 minutes to prepare a total volume of 100 μl, 20 μl 10 × second strand buffer, 6 μl second strand dNTP mixture, and 61 μl sterile water were added. 2 μl RNase H (1.5 U / μl) and 11 μl DNA polymerase I (9.0 U / μl) were further added, followed by reaction at 16 ° C. for 2 hours and 30 minutes, and immediately put on ice. 23 μl blunting dNTP mix and 2 μl cloned Pfu DNA polymerase (2.5 U / μl) were added to the second strand synthesis product, and then rapidly vortexed and reacted at 72 ° C. for 30 minutes. Purification using a QIAquick® PCR Purification Kit (final volume 50 μl).

③ Pyro Sequencing (GS FLX Titanium Run)

Using the synthesized cDNA, a DNA library was prepared and subjected to a PCR procedure (clonal amplification) to attach DNA Capture Beads. The sequencing run sequentially flowed nucleotides into a platform called PicoTiterPlate with millions of wells, and when the nucleotides flowed through PicoTiterPlate, sequencing analysis was performed on sequencing beads with millions of DNA fragments on the surface where clonal amplification occurred. This is done. At this time, if nucleotides complementary to the dNTPs flowing on the surface of the sequencing bead are synthesized by the polymerase, at the same time, light is generated in the synthesized wells, and the sequence of the EST is detected using data after being detected by the CCD camera. Could get

Example  2. SOLEXA  Sequencing ( SOLEXA seqeuncing )through Fullerene  Acquisition of specific expression genome database according to processing

Funnels with fullerene treatment ( Chironomus To obtain a specific expression genome database (DB) of riparius ), 50 rats of 4 stage larvae treated with 1 mg / L fullerene at 20 ° C. for 24 hours and untreated controls, David et al. (David et al. BMC Genomics 2010 11: 216), SOLEXA sequencing was performed. The entire genome list obtained from the previously performed pyro sequencing was examined for the expression of genes due to fullerene exposure.

As shown in FIG. 1, the expression level of the genome was determined by measuring the number at a predetermined portion of the sequence during sequencing. Through this, genes increased or decreased by fullerene were distinguished. Among the 25% of genomes for which genetic information can be obtained, only the genomes showing the tendency to increase or decrease in the treatment group were classified into ontology as shown in FIG. 2. . Confirmation list of fullerene treatment group DEG (Differential Expressed Genes) is shown in Table 1. By comparing Highest Depth values, we selected 72 genes that were meaningful in virulence mechanisms. The cDNA sequence database obtained through sequencing into a file is then converted to another species in the NCBI database or the previously identified Chironomus by the BLASTx method. The accessibility of riparius with its amino acid sequence was confirmed and listed. NCBI blast X search in the database to classify contig with genomic information, of which the sequence length is 500 bp or more, and the list of Highest Depth values of 100 or more, which is considered to have high expression in this species in the treatment group. I figured out. Among the contig showing more than 10% increase and decrease, genes commonly used as stress markers were selected, and among them, 72 genes which can be mainly identified in similar insects were selected as biomarkers.

Name Highest depth up - down ratio Gene Chiro -
control Chiro - C60 contig00006
(SEQ ID NO: 1) 11131 2015 -82% CHK1 checkpoint-like protein
Helicoverpa armigera contig00447
(SEQ ID NO: 2) 980 1490 52% Metallothionein family protein
[Trichomonas vaginalis G3] contig00462
(SEQ ID NO: 3) 2152 3529 64% heat shock cognate 70
[Chironomus yoshimatsui] contig00726
(SEQ ID NO: 4) 5031 4154 -17% ribosomal protein S3
[Chironomus riparius] contig00772
(SEQ ID NO: 5) 214 321 50% heat shock transcription factor (hsf) [Aedes aegypti] contig00835
(SEQ ID NO: 6) 4681 3105 -34% cytochrome b [Culicoides arakawae] contig00840
(SEQ ID NO: 7) 2284 1903 -17% 60S ribosomal protein L7a
[Culex quinquefasciatus] contig00849
(SEQ ID NO: 8) 194 307 58% ATP-citrate synthase [Aedes aegypti] contig00936
(SEQ ID NO: 9) 200 317 59% acyl-CoA oxidase [Culex quinquefasciatus] contig01163
(SEQ ID NO: 10) 3252 4903 51% 40S ribosomal protein S7 [Aedes aegypti] contig01191
(SEQ ID NO: 11) 1280 1528 19% map kinase-interacting serine / threonine kinase [Aedes aegypti] contig01263
(SEQ ID NO: 12) 516 711 38% aldehyde dehydrogenase [Aedes aegypti] contig01343
(SEQ ID NO: 13) 180 44 -76% hemolymph proteinase 19 [Manduca sexta] contig01678
(SEQ ID NO: 14) 10680 13619 28% actin 1 [Nilaparvata lugens] contig02823
(SEQ ID NO: 15) 246 359 46% nucleolar GTP-binding protein
[Aedes aegypti] contig03291
(SEQ ID NO: 16) 2335 1021 -56% larval serum protein 2
[Culex quinquefasciatus] contig03359
(SEQ ID NO: 17) 7389 6076 -18% ribosomal protein Ubq / L40e
[Agriotes lineatus] contig03624
(SEQ ID NO: 18) 1406 1927 37% glutamate dehydrogenase, short peptide [Drosophila melanogaster] contig03983
(SEQ ID NO: 19) 195 335 72% cytochrome P450 [Culex quinquefasciatus] contig04024
(SEQ ID NO: 20) 533 778 46% mitochondrial malate dehydrogenase 2
[Culex quinquefasciatus] contig04162
(SEQ ID NO: 21) 580 795 37% utp-glucose-1-phosphate uridylyltransferase 2 [Culex quinquefasciatus] contig04618
(SEQ ID NO: 22) 6116 3720 -39% NADH dehydrogenase subunit 1
[Anopheles gambiae] contig04622
(SEQ ID NO: 23) 693 453 -35% DNA-binding nuclear protein p8
[Simulium vittatum] contig05549
(SEQ ID NO: 24) 265 422 59% hemolymph proteinase 6 [Manduca sexta] contig06416
(SEQ ID NO: 25) 253 777 207% cytochrome P450 [Aedes aegypti] contig06433
(SEQ ID NO: 26) 477 945 98% glucose-6-phosphate isomerase
[Culex quinquefasciatus] contig06748
(SEQ ID NO: 27) 543 999 84% carboxylesterase [Aedes aegypti] contig07318
(SEQ ID NO: 28) 129 275 113% alcohol dehydrogenase 3
[Chironomus riparius] contig07720
(SEQ ID NO 29) 185 314 70% glutathione S-transferase
[Chironomus tentans] contig07984
(SEQ ID NO: 30) 789 1175 49% mitochondrial NADH: ubiquinone oxidoreductase NDUFV1 / 51 kDa subunit [Aedes aegypti] contig08276
(SEQ ID NO 31) 264 155 -41% glutathione transferase, zeta class (AGAP002898-PA) [Anopheles gambiae str. PEST] contig08697
(SEQ ID NO 32) 248 351 42% aldehyde dehydrogenase [Aedes aegypti] contig09526
(SEQ ID NO: 33) 514 859 67% DNA polymerase [Fowlpox virus] contig10025
(SEQ ID NO 34) 409 554 35% mitochondrial processing peptidase beta subunit [Aedes aegypti] contig10185
(SEQ ID NO: 35) 762 1177 54% 60S ribosomal protein L24 [Aedes aegypti] contig11219
(SEQ ID NO: 36) 429 566 32% vitellogenin [Culex quinquefasciatus] contig13104
(SEQ ID NO: 37) 547 945 73% NADH-ubiquinone oxidoreductase sgdh subunit [Culex quinquefasciatus] contig14290
(SEQ ID NO: 38) 3656 9326 155% hemoglobin 13 [Chironomus thummi] contig20375
(SEQ ID NO: 39) 548 682 24% heat shock protein [Aedes aegypti] contig20379
(SEQ ID NO: 40) 1175 1589 35% cytochrome c oxidase subunit VIIa
[Culex quinquefasciatus] contig20383
(SEQ ID NO: 41) 420 306 -27% cytochrome p450 [Aedes aegypti] contig20408
(SEQ ID NO: 42) 875 1162 33% hemoglobin CTT6 [Polypedilum vanderplanki] contig20500
(SEQ ID NO: 43) 108 396 267% Metallothionein family protein
[Trichomonas vaginalis G3] contig20537
(SEQ ID NO: 44) 2724 3923 44% ribosomal protein S3 [Aedes albopictus] contig20580
(SEQ ID NO: 45) 1222 1544 26% hemoglobin CTT6 [Polypedilum vanderplanki] contig20591
(SEQ ID NO: 46) 543 194 -64% pupal cuticle protein, putative
[Aedes aegypti] contig20678
(SEQ ID NO: 47) 2338 2915 25% ribosomal protein L35
[Culicoides sonorensis] contig20781
(SEQ ID NO 48) 3244 3608 11% ribosomal protein L18a
[Polypedilum vanderplanki] contig20843
(SEQ ID NO: 49) 1944 2489 28% ribosomal protein L3 [Aedes aegypti] contig20854
(SEQ ID NO: 50) 1811 1460 -19% 40S ribosomal protein S24 [Aedes aegypti] contig20895
(SEQ ID NO: 51) 750 585 -22% mitochondrial ATP synthase F chain [Ochlerotatus triseriatus] contig20923
(SEQ ID NO: 52) 972 1157 19% multifunctional chaperone
[Simulium nigrimanum] contig20931
(SEQ ID NO: 53) 2341 1667 -29% 60S ribosomal protein L6 [Aedes aegypti] contig20956
(SEQ ID NO: 54) 499 764 53% alcohol dehydrogenase 3
[Chironomus riparius] contig21096
(SEQ ID NO: 55) 2704 3817 41% heat shock cognate 70 [Chironomus tentans] contig21110
(SEQ ID NO: 56) 2166 4007 85% calcium-transporting atpase sarcoplasmic / endoplasmic reticulum type (calcium pump) [Aedes aegypti] contig21114
(SEQ ID NO: 57) 6822 9858 45% 60S ribosomal protein L8 [Culex tarsalis] contig21172
(SEQ ID NO: 58) 8341 871 -90% putative senescence-associated protein [Cupressus sempervirens] contig21194
(SEQ ID NO: 59) 3221 3940 22% cytochrome b [Chironomus inquinatus] contig21199
(SEQ ID NO: 60) 422 313 -26% heat shock cognate 70 [Aedes aegypti] contig21217
(SEQ ID NO: 61) 811 979 21% cytochrome C oxidase subunit IV-like protein [Phlebotomus papatasi] contig21230
(SEQ ID NO: 62) 619 278 -55% zinc carboxypeptidase [Aedes aegypti] contig21269
(SEQ ID NO: 63) 6079 8711 43% putative ribosomal protein S4e [Graphocephala atropunctata] contig21299
(SEQ ID NO: 64) 7022 4576 -35% cytochrome oxidase subunit II
[Chironomus calligraphus] contig21301
(SEQ ID NO: 65) 3372 3876 15% 40S ribosomal protein S11
[Culex quinquefasciatus] contig21315
(SEQ ID NO: 66) 7786 9238 19% ribosomal protein rps23 [Arenicola marina] contig21328
(SEQ ID NO: 67) 1437 1700 18% heat shock cognate 70 [Chironomus tentans] contig21482
(SEQ ID NO: 68) 3864 2604 -33% ribosomal protein L9 [Culicoides sonorensis] contig21542
(SEQ ID NO: 69) 898 1124 25% aldehyde dehydrogenase
[Culex quinquefasciatus] contig21550
(SEQ ID NO: 70) 1114 1436 29% malate dehydrogenase
[Culex quinquefasciatus] contig21554
(SEQ ID NO: 71) 784 1096 40% alcohol dehydrogenase 3
[Chironomus riparius] contig21823
(SEQ ID NO: 72) 2751 1743 -37% 60S ribosomal protein L22
[Culex quinquefasciatus]

Attach an electronic file to a sequence list

Claims (6)

delete Gene chip for evaluation of ecotoxicity of fullerene (fullerene) comprising a gene derived from Chinono s riparius consisting of the nucleotide sequence of SEQ ID NO: 1 to 72. Kit for evaluating the ecotoxicity of fullerene (fullerene) comprising a gene derived from Chihironomus riparius consisting of the nucleotide sequence of SEQ ID NO: 1 to 72. Exposing the funnel to a sample suspected of contaminated with fullerene and isolating RNA therefrom;
Synthesizing cDNA using the separated RNA as a template;
Contacting the synthesized cDNA with the gene chip according to claim 2 and hybridizing with the cDNA;
Detecting a degree of hybridization between the gene chip and cDNA; And
Comparing the detected degree of hybridization with a positive control group. 5. The method of claim 4, wherein the cDNA is labeled with a detectable labeling substance. Composition for evaluating ecotoxicity of fullerene (fullerene) comprising a gene derived from Chinonomus riparius consisting of the nucleotide sequence of SEQ ID NO: 1 to 72.

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