CN106831973B

CN106831973B - Water estrogen pollution detection method based on mackerel vitellogenin

Info

Publication number: CN106831973B
Application number: CN201710163953.3A
Authority: CN
Inventors: 吴美琴; 陈渊戈; 葛珂珂; 练青平
Original assignee: Ninghai County Sanmenwan District Modern Fishery Park Administrative Committee; Shanghai Ocean University; East China Sea Fishery Research Institute Chinese Academy of Fishery Sciences; Zhejiang Institute of Freshwater Fisheries
Current assignee: Ninghai County Sanmenwan District Modern Fishery Park Administrative Committee; Shanghai Ocean University; East China Sea Fishery Research Institute Chinese Academy of Fishery Sciences; Zhejiang Institute of Freshwater Fisheries
Priority date: 2017-03-17
Filing date: 2017-03-17
Publication date: 2020-10-16
Anticipated expiration: 2037-03-17
Also published as: CN106831973A

Abstract

The invention discloses a method for detecting estrogen pollution of water body based on vitellogenin of mudskipper, belonging to the field of molecular biology and environmental pollution detection; the amino acid sequence of the mackerel vitellogenin is shown as SEQ ID N0.2. At present, no mudflat fish is used for biological in-vivo detection of environmental estrogen, and the corresponding biological detection technology is lacked. The invention selects the mudflat settleable and environmentally sensitive mudskipper periodophallus modestus living model, designs the primer, adopts the real-time quantitative fluorescence PCR method to detect the expression of the vitellogenin VtgAa, and the detection method can be used for detecting the environmental estrogen pollution degree of water areas such as seawater, river estuary and the like.

Description

Water estrogen pollution detection method based on mackerel vitellogenin

Technical Field

The invention belongs to the field of molecular biology and environmental pollution detection, and particularly relates to a water body environmental estrogen detection method based on novel mudflat model fish-mudskipper vitellogenin (vtg), which can be widely applied to the assessment of environmental estrogen pollution degree of a estuary mudflat water area.

Background

In the modern generation of the rapid development of the industry, a large amount of chemical substances are unreasonably released in the environment, and the modern agriculture is excessively dependent on pesticides, so that the environment is greatly polluted. There is increasing evidence that there are a variety of substances in the environment that mimic and interfere with endocrine function in animals and humans. After entering the body, these exogenous compounds interfere with the processes of synthesis, release, transportation, metabolism, combination and the like of endocrine of the body, and thus cause the change of sex and deformity of animals. This large class of substances is known as Environmental Endocrine Disruptors (EEDs), Environmental estrogens (Environmental estrogens), or Endocrine active compounds (Endocrine compounds). The environmental estrogen problem is compared with the ozone layer destruction and the greenhouse effect, and the attention of the environmental estrogen problem is paid to the environmental estrogen problem.

Most of the current research work on endocrine disruptors is still directed to the simulated exposure of a single compound, and the actual environmental system contains a series of substances with different estrogenic activities. Therefore, accurate assessment of ecological risk is difficult based on toxicological data of individual substances. The estrogenic activity of the mixture was studied to be more consistent with the conditions existing in the actual environment than the individual substances. The mixture of several estrogen-like substances in concentrations that are not effective has significant estrogenic activity. The establishment of an economical, convenient and sensitive method for detecting the activity of the environmental estrogen mixture, particularly the search for efficient, specific and sensitive biomarkers, has been the focus of research on environmental endocrine disrupting substances.

Environmental estrogens are various in variety, chemical analysis cannot refine all pollutants with estrogen effect in environmental samples, and the intensity of toxic effect cannot be known only from the concentration of environmental estrogens. Thirdly, the chemical analysis method has high dependence on the instrument and is time-consuming and labor-consuming. The application of the biomarker well solves the defect, the detailed compound composition of the sample is not required to be known by measuring the whole endocrine disrupting effect of the sample, and the advantages of simple operation, rapidness, economy and high efficiency are provided, so that the biomarker becomes a main means for monitoring the environmental endocrine disrupting chemicals.

Periophthalmus modestus belongs to the order Perciformes, the suborder gobies, the family Botrytidae and the genus Botrytis, inhabits in the estuary saline-fresh water area, the near-shore mudflat position or the low-tide region of bottom mud, and is a coastal warm water wide-temperature wide-salinity fish.

Vitellogenin (Vtg) of fish is a yolk precursor protein which is synthesized by liver under the action of estradiol (estradiol-17 beta, E2) and secreted into blood. Normally, Vtg is a female specific serum protein present in the blood of female fish during vitellogenesis, but male and young fish can also induce Vtg production in vivo under the action of exogenous estrogen. According to this characteristic, Vtg can be used as an indicator protein for judging sex and maturity of female fish, and the contamination of exogenous estrogen substances (environmental estrogen) in the water environment can also be evaluated by detecting abnormal generation of Vtg in male fish or young fish (Specker & Sullivan, 1994). At present, a part of vitellogenin has developed an estrogen-like pollution detection method at home and abroad, but the selected fish model is mainly freshwater cyprinid fish, and no patent report on the aspect of application of mudflat water fish in environmental estrogen detection is found. The freshwater carps and the mudskipper live in different water bodies, and the application of the mudskipper to the environmental estrogen detection has irreplaceable practical significance for the carps due to the particularity of the habitat of the mudskipper. The research selects mudflat colonized mudskipper as a living body model, establishes a living body environment detection technology aiming at the vitellogenin, and has higher innovation and practicability.

Disclosure of Invention

The invention aims to provide a method for detecting estrogen pollution of water body based on the vitellogenin of the mudskipper, which is applied to the environmental estrogen pollution detection of each water area.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention relates to the use of mackerel vitellogenin as an indicator protein for detecting environmental estrogen pollution, wherein the amino acid sequence of the mackerel vitellogenin is shown as SEQ ID No. 2.

Preferably, the nucleotide sequence of the mudskipper VtgAa gene for coding the yolk proteinogen of the mudskipper is shown as 3 rd to 5000 th positions in SEQ ID NO. 1.

In a second aspect, the present invention relates to a test method for detecting contamination of an aquatic environment, the method comprising detecting the expression level of mackerel vitellogenin in a fish in the aquatic environment, the amino acid sequence of the mackerel vitellogenin being represented by SEQ ID No. 2.

Preferably, the aquatic environment pollution is an estrogenic effect.

Preferably, the method comprises the steps of:

s1, extracting total RNA from the liver tissue of the young or the male mudfish, and carrying out reverse transcription amplification;

s2, designing VtgAa and internal reference ef 1-alpha gene fluorescent quantitative PCR primers respectively, and establishing an absolute quantitative PCR method;

s3, quantifying the mRNA expression level of the vitellogenin VtgAa in the sample, and analyzing the environmental estrogen pollution degree of the sample.

In a third aspect, the present invention relates to a method for cloning the full-length sequence of the mudskipper VtgAa gene, comprising the steps of:

a1, extracting total RNA from the liver tissue of the female mudskipper in the sexual maturity stage, and carrying out reverse transcription amplification;

a2, designing a degenerate primer of VtgAa to carry out amplification reaction to obtain a VtgAa gene fragment;

a3, respectively preparing cDNA sequences of VtgAa gene 3 'end and 5' end by using total RNA of the mudskipper as a template according to a RACE method;

a4, designing a specific full-length primer, and amplifying to obtain a full-length sequence of the VtgAa gene.

Preferably, in the step A2, the degenerate primers corresponding to the VtgAa gene are vtgAa-degenerate-F1 shown in SEQ ID NO.3 and vtgAa-degenerate-R1 shown in SEQ ID NO. 4.

Preferably, in step A3, the RACE primers corresponding to VtgAa gene are vtgAa-5'RACE-GSP1 shown in SEQ ID NO.7 and vtgAa-3' RACE-GSP2 shown in SEQ ID NO. 8.

Preferably, in step A4, the specific full-length primers corresponding to the VtgAa gene are vtgAa-full primer-F1 shown in SEQ ID NO.11 and vtgAa-full primer-R1 shown in SEQ ID NO. 12.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention obtains full-length CDS sequence of the mudskipper VtgAa gene and fills the blank of the mudskipper Vtg gene family.

2. The mudskipper is common estuary fish widely distributed in the southeast coast of China, Vtg is an important biomarker for understanding environmental estrogen pollution of low-tide regions of estuary saline and fresh water areas, near-shore mud flats or bottom mud, the full length of VtgAa gene is obtained, and the mudskipper has important significance for more accurately indicating the pollution condition of corresponding water areas and more deeply exploring the problems of protein expression and the like of aquatic organisms in the polluted water areas.

3. The invention establishes a real-time fluorescent quantitative PCR method of the mudskipper VtgAa and ef 1-alpha (internal reference) genes by designing specific primers. The change of the expression level of VtgAa under estrogen induction is detected by a fluorescent quantitative PCR method, so that the estrogen pollution condition in the environment is indicated.

Drawings

FIG. 1 is a full-length electrophoresis diagram of cDNA of the vitellogenin VtgAa and ef 1-alpha genes of Pacific salmon; wherein, M: marker, 1: VtgAa, 2: ef 1-alpha;

FIG. 2 is a standard graph of fluorescent quantitative PCR of the Pacific carp VtgAa gene and the reference gene ef 1-alpha;

FIG. 3 shows the variation of VtgAa expression levels induced by different estrogen concentrations over time.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

Examples

The embodiment relates to a vitellogenin (vtg) gene of a mudskipper, a coded protein, application thereof and a method for cloning a cDNA full-length sequence.

VtgAa gene: the mudskipper VtgAa gene is characterized in that the total length of a VtgAa sequence is 5223bp, and the vtgAa sequence comprises an initiation codon ATG, a termination codon TAG and polyA. The Open Reading Frame (ORF) of the gene is located at 3-5000 bp, is 4998bp long, and encodes 1665 amino acids, wherein the signal peptide is 15 amino acids. According to calculation, the molecular weight of the encoded protein is 185 kDa. The coding amino acid sequence has a typical complete vitellogenin structure: 1 vitellogenin domain (located in 24-594 amino acids of the coded amino acid peptide chain), 1 VWFD domain (located in 1423-1555 amino acids of the coded amino acid peptide chain), 1 polyserine domain (located in 1064-1195 amino acids of the coded amino acid peptide chain), and 1 proteolytic cleavage site (RSRR). The deduced amino acid sequence was aligned with other fish Vtg amino acid sequences and found to contain 4 portions of LvH, LvL, Pv and β components.

The nucleotide sequence of the mudskipper VtgAa gene is shown in SEQ ID NO. 1.

The coding region of the obtained full-length VtgAa cDNA sequence is from nucleotide 3 to nucleotide 5000.

The amino acid sequence of the mudskipper VtgAa gene is shown in SEQ ID NO. 2.

The invention establishes a real-time fluorescent quantitative PCR method for the VtgAa and ef 1-alpha (internal reference) genes of the mudskipper by designing specific primers which are shown as SEQ ID NO. 15-18. The expression change of the VtgAa gene under the induction of estrogen with different concentrations is detected by a fluorescent quantitative PCR method, and the result shows that: the induction effect is obviously enhanced along with the increase of the concentration; there was a clear time-dependent effect of VtgAa expression (same concentration) with increasing exposure time.

Method for cloning the full-length sequence of cDNA: extracting total RNA from liver tissues of female mudskipper in the sexual maturity stage, carrying out reverse transcription amplification, designing a degenerate primer of Vtg, and obtaining a corresponding gene segment; on the basis, SMART RACE technology is adopted to obtain 5 'and 3' end unknown sequences; finally, designing full-length upstream and downstream primers, and cloning to obtain a corresponding full-length cDNA sequence. The complete Open Reading Frame (ORF) was confirmed and its individual domains were analyzed using Macvector software. And establishing a system analysis chart by aligning complete amino acid sequences, and determining the Vtg type of the protein.

In the implementation process, the technical scheme for cloning the full-length cDNA sequence can adopt the following specific steps:

1. collecting animal tissue and organ samples:

collecting liver of fresh female fish of Pacific Oncorhynchus Linnaeus, cutting, and storing in RNAlater for total RNA extraction.

2. Extraction and reverse transcription amplification of total RNA of mudskipper

Extracting total RNA according to the ISOGEN II kit operation instruction, wherein the specific method comprises the following steps:

(1) placing liver tissue (about 100mg) into 1.5ml centrifuge tube, adding 1ml ISOGEN II, homogenizing with electric homogenizing rod, adding 0.4ml RNase free water, shaking vigorously for 15s, and standing at room temperature for 10 min; centrifugation was carried out at 4 ℃ for 10min at 12 Kxg.

(2) Taking 1ml of the supernatant, adding 5 mul of p-bromoanisole, shaking vigorously for 15s, and standing for 3min at room temperature; centrifugation was carried out at 4 ℃ for 10min at 12 Kxg.

(3) Taking 1ml of the supernatant, adding 1ml (equal volume) of isopropanol, reversing, uniformly mixing, and standing at room temperature for 10 min; centrifuging at 4 deg.C for 10min at 12 Kxg to obtain white precipitate at the bottom and wall of the tube as RNA precipitate.

(4) Discarding the supernatant, adding 0.5ml of 75% ethanol into the RNA precipitate to wash the precipitate, centrifuging at 4 ℃ for 3min at 7 Kxg, and discarding the supernatant; this step is repeated once.

(5) Drying the precipitate for 2min in shade at room temperature.

(6) Adding 50 μ l (or proper amount) of RNase free water to dissolve the precipitate, and the solution is the total RNA solution.

(7) Total RNA integrity was checked by 0.8% agarose TAE gel electrophoresis; total RNA concentration and purity were determined using a Thermo Fisher NanoDrop2000 spectrophotometer.

(8) Mixing the above total RNA

VILO^TMcDNA Synthesis Kit (Invitrogen) was reverse transcribed into cDNA template.

Note: the above-mentioned centrifuge tubes, homogenizing rods, etc. were all subjected to RNase free treatment.

2. Degenerate primers amplify Vtg gene and ef 1-alpha reference gene fragments

The Vtg gene genus has conservative sequence, a pair of degenerate primers are designed according to the conserved sequence of the Vtg gene based on CDS sequence of halibut, Pagrus major, Mugillus bardadae, Musca fascicularis and goby, and a pair of specific primers are designed according to the conserved sequence of ef 1-alpha gene, wherein the sequence is as follows:

vtgAa-degenerate-F1	5’-AARATGAAGCGYRTCMTDMAGGCTCT-3’SEQ ID NO.3
		vtgAa-degenerate-R1	5’-AACWGGMAGRRCTYSDAKCTTAAAST-3’SEQ ID NO.4
ef1-α-F1	5’GCCCTGCTGGCCTACACCCT--3’SEQ ID NO.5
		ef1-α-R1	5’-GGTGGTTCAGGATGATGACCT-3’SEQ ID NO.6

the PCR amplification reaction system is a 10-microliter system: mu.l each of F and R-terminal primers (10. mu.M), 0.5. mu.l of cDNA template, ddH₂O2.5. mu.l, Mastermix 5. mu.l, at 95 ℃ for 5min, 35 cycles (95 ℃, 30 s; 54 ℃, 30 s; 72 ℃, 1min), and extension at 72 ℃ for 10 min.

4. Recovery and purification of PCR products

Mu.l of the PCR reaction was subjected to 1.5% agarose gel electrophoresis to confirm the intermediate sequence amplification product. The band of interest product was recovered by purification with GENECLEAN Turbo Kit (MPBiomecicals Europe, France) tapping.

5. Ligation transformation of PCR amplified fragments of interest

(1) Connecting: mu.l of the PCR product, 1. mu.l of pGEM-T easy vector (Promega, USA), 1. mu.l of T4ligase, and 5. mu.l of Buffer were added to a 0.2ml PCR tube, mixed gently, and then allowed to stand overnight at 4 ℃.

(2) And (3) transformation: adding 5 μ l of the ligation product into competent cells of Escherichia coli DH5 alpha, flicking and mixing uniformly, and carrying out ice bath for 30 min; heat shock at 42 deg.C for 45s, and immediately placing on ice for 2 min; plates were smeared and the dishes were placed upside down in a 37 ℃ incubator overnight.

6. Identification, sequencing and sequence analysis of positive clones

To a 0.2ml PCR tube, 10ul of the following mix was added: AmpliTaq

MasterMix 5 mul, T7 and SP6 primers (10 mul) 0.5 mul each, sterile water 4ul, using sterile toothpick to take single clone, putting it into the PCR tube, making it fully contact, and making colony PCR verification, the PCR reaction conditions are pre-denaturation at 95 deg.C for 5min, 35 cycles (denaturation at 95 deg.C for 30s, annealing at 50 deg.C for 30s, and extension at 72 deg.C for 1min), extension at 72 deg.C for 10min, confirming clone containing target fragment, enlarging culture, sequencing with T7 and SP6 primers, 579bp VtgAa and 640bp ef1- α gene fragments are obtained.

7.5 'RACE and 3' RACE amplification of Photorhabdus fasciatus Vtg5 'and 3' end gene sequences

The RACE primers were designed using Macvector as follows:

vtgAa-5'RACE-GSP1	5’-CCTGGACCTTGGCTCTTGAGACTATGG-3’SEQ ID NO.7
		vtgAa-3'RACE-GSP2	5’-GTCACTTGTTCCAAAATGGTGTCTCCGC-3’SEQ ID NO.8
ef1-α-5'RACE–GSP1	5’-CGTTTCCACGACGGATTTCCTTG-3’SEQ ID NO.9
		ef1-α-3'RACE-GSP2	5’-TGTCCTGGTTCAAGGGATGGAAG-3’SEQ ID NO.10

according to

RACE 5 '/3' kit (Clontech) instructions, using total Pacific Oncorhynchus RNA as a template to prepare 3 'end and 5' end cDNA, and preparing a PCR reaction solution with specific primers (GSP) to a total volume of 25. mu.l, including 2.5. mu.l 10 × Advantage 2PCRbuffer, 0.5. mu.l dNTPs mix (10mM), 0.5. mu.l GSP (10. mu.M), 2.5. mu.l 10 × UPM, 1.25. mu.l template, and 0.5. mu.l 10 × Advantage 2polymerase mix (Clontech). The PCR reaction conditions are 5 cycles (94 ℃, 30 s; 72 ℃, 3min), 5 cycles (94 ℃, 30 s; 70 ℃, 30 s; 72 ℃, 3min), 25 cycles (94 ℃, 30 s; 68 ℃, 30 s; 72 ℃, 3 min).

The ligation transformation procedure after PCR reaction was the same as 5 for 5'RACE and 3' RACE.

8. Amplification of the full Length VtgAa Gene

According to the cDNA sequences of the 3 'end and the 5' end obtained by the RACE method, specific full-length primers are respectively designed as follows:

vtgAa-fullprimer-F1	5’-CAGCCATGAGAGTCGTTGTACTAGCCT-3’SEQ ID NO.11
		vtgAa-fullprimer-R1	5’-CAAGCAGTGGTATCAGCGCAGAGTA-3’SEQ ID NO.12
ef1-α-fullprimer-F1	5’-GCCTTGAAAAACCCAGAAACACCG-3’SEQ ID NO.13
		ef1-α-fullprimer-R1	5’-GGTGATGTGAGGGGAAAGGGAG-3’SEQ ID NO.14

the following PCR reaction solution was prepared: including 10 × Advantage 2PCRbuffer (5 μ l), dNTPs mix (10mM,1 μ l), upstream and downstream primers GSP (10 μ M,1 μ l each), template (1 μ l) and 10 × Advantage 2polymerase mix (1 μ l), plus N.F.W. to a total reaction volume of 50 μ l. The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 1 min; 5 cycles (95 ℃, 30 s; 68 ℃, 3min), 25 cycles (94 ℃, 30 s; 68 ℃, 30 s; 72 ℃, 3min), 68 ℃ extension for 3 min.

The PCR product was detected by 1% agarose gel electrophoresis for amplification effect as shown in FIG. 1. As can be seen from FIG. 1, the expected VtgAa band with a total length of about 5000bp was obtained by amplification with the primers SEQ ID19 and SEQ ID20, respectively, using cDNA as a template; the expected ef 1-alpha band with the full length of about 1500bp is obtained by amplification with primers SEQID23 and SEQID 24. Wherein, M: marker, 1: VtgAa, 2: ef 1-alpha.

9. Estrogen induction effect test

(1) The test fish is collected from a natural pollution-free water area of a wetland in east beach of Shanghai Chongming island, young fish is temporarily cultured in 10L glass water tanks, the water temperature is 18, the pH value is 7.5, and water is changed once a day.

(2) Test inducer: endogenous estrogen estradiol (E2); cosolvent: anhydrous ethanol.

(3) Induction test: the induction experiment of the mackerel vitellogenin is implemented by adopting a method of exposing and dissolving estrogen in water. The experiment was divided into E2 group, control group, and each treatment was divided into 2 replicates. Group E2 was set to 3 exposure concentrations (10ng/L, 100ng/L, 1000 ng/L). The control components are blank control and original control, wherein the water body of the blank control is added with cosolvent absolute ethyl alcohol with equal dosage, and the original control is not added with any reagent. At 0, 2 and 7 days after exposure, each treatment group was sampled randomly, 5 tails per group, and liver tissue was harvested from the biopsies after anesthesia, minced and stored in RNAlater storage for later use.

10. Sample processing

Extracting total RNA of liver by ISOGEN II kit, identifying RNA quality and detecting RNA concentration by spectrophotometer, collecting 1 μ g RNA, and reverse transcription kit of Invitrogen company

VILO^TMThe cDNA Synthesis Kit was reverse transcribed to cDNA, 10. mu.l reaction containing 2. mu.l of 5XVILO^TMReaction Mix,1μl 10X

enzymeMix, 1. mu.g RNA and DEPC-treatedwater. The reaction procedure is as follows: at 25 ℃ for 10 min; 60min at 42 ℃; 85 ℃ for 5 min. First strand cDNA was obtained by reverse transcription and used in the next experiment.

11. Establishment of real-time fluorescent quantitative PCR method

Recombinant plasmids containing VtgAa and ef1- α (internal reference) gene fragments were extracted from E.coli, respectively, and their DNA concentration and purity were measured on a spectrophotometer to prepare plasmid cDNAs (target gene and internal reference)Ginseng gene) as template, diluting to 10¹To 10⁶ Copy number 6 gradients, each gradient 2 replicates, a standard curve (FIG. 2) was made for the gene Vtg and ef1- α (internal reference) the actual absolute quantification of the gene of interest was calculated from the standard curve.

Real-time fluorescent quantitative PCR reaction A20. mu.l reaction system was configured using Power SYBR Green PCRMasterMix (applied biosystems, USA): mu.l cDNA, F andRpirimer (final concentration 200nM), SYBR GreenMix (10. mu.l), N.F.W. A no reverse transcription sample control and an inter-group control were performed simultaneously. The PCR reaction conditions are as follows: 1 cycle (50 ℃, 2 min; 95 ℃,10min), 40 cycles (95 ℃,15 s; 60 ℃,1 min).

Based on the obtained full-length cDNA sequence of the vitellogenin VtgAa, the following primers were designed for real-time fluorescent quantitative PCR reaction using Primer3Plus (http:// www.bioinformatics.nl/cgi-bin/Primer 3Plus. cgi /).

ef1-αqPCR-F1	5’-TGGTGACAGCAAGAACAACC-3’SEQ ID NO.15
		ef1-αqPCR-R1	5’-ATGAACTTGCAGGCGATGTG-3’SEQ ID NO.16
vtgAa-qPCR-F1	5’-AAATTGCCAGAGAGCGCTTG-3’SEQ ID NO.17
		vtgAa-qPCR-R1	5’-AACAGCTTGCCGTTCATCAG-3’SEQ ID NO.18

12. Estrogen induction results of VtgAa Gene

As shown in FIG. 3, the expression of VtgAa gene in E2 exposure was detected by real-time fluorescent quantitative PCR, and ef 1-alpha gene was used as a control gene, and was expressed in the exposed group, but not in the control group. The expression is carried out at a low concentration of 10ng/L, and the induction effect is obviously enhanced along with the increase of the concentration; from the exposure time point of view, each concentration exposure group has obvious time-dependent effect.

SEQUENCE LISTING

<110> Shanghai ocean university

<120> water body estrogen pollution detection method based on mudskipper vitellogenin

<130>2017

<160>18

<170>PatentIn version 3.5

<210>1

<211>5223

<212>DNA

<213>Periophthalmus modestus（VtgAa）

<400>1

acatggggac aacccactca gccatgagag tcgttgtact agccttggct ctagcccttg 60

tggctggcca gcacgataac ttggctccta gttttgctcc cggactaacc tatgtgtaca 120

agtacaatgc ccagagcctg ggaggtctgt ctgaacagca cctagctaaa gccggactca 180

acttcaccag caatgtcaag atcagtgttg cccaagaaaa cgtcctcatg cttcagcttg 240

agaatcctcg gatctatgag ttcagtggtg tttggccaaa ggattcctac gtacaaactc 300

acctccgtgc cgacctggaa gctcatctca aaaccgccat caagttcaaa tatgaccatg 360

ggattgtgag agagatcctg gccccagaga gtgtccccat actgctgctc aacatcttta 420

gaggcatcct caacttcttc cagctgaaca tcaagaagtc acagaatgtt tatgaactgc 480

aggaggaggg agcccagggc gtgtgcaaga cccagtatgc catcacagag aacgataagg 540

ctgagcgcat ccttttgacc aagagcagga acttgaatca ctgccaggag aaggtcatga 600

gggatatcgg gttggcatac actaagacat ggcataagtg ccgggagatt tccaaaaacc 660

tgaggggaac cacaggatac ttctacaagc tgaaggcagc cccaggcggc ttgatcattg 720

agaaggcctc cggaaaggaa gtcattcagt tcacaccttt taatgaccag aatggtgctg 780

cttctatgca gacaatccaa accctggatt tcattgctgc catcaaggct cctattgttc 840

ccatcagtgc tccgtaccat ccacgtggct ccctgaaata tcagttttcc actgagcttc 900

tgcagagccc cctcaggatc cttaagatgt cagacgtgaa ggaacaggtt gctgatgttc 960

tgaacaacct ggttgtcaat aacagagaca aagtacatga ggatgctcct ctcaagtttt 1020

ttgagctgat cctgctgctg cgtgcttctg atttgactga actgcgcaac ctgctgacta 1080

cctacaaaag cagacctctt gagaggcggt ggttgatgga cgccatcagc aacactggaa 1140

caaaggctgc tttggagatt gtcatggctg agatccagaa gagagagcta tctgttcctg 1200

aagccgctca agttttgatt ggaactctgc acatgttgaa gcccactgac gagatcatcc 1260

agaaggtttg gagctacatt gagcagctct cacaggaaca aggacgctac gaacaagttg 1320

tgcgcaaagc tctgttcctg ggctatggta gcatcatcca cagacagagt gttcagaggg 1380

ctgagtggaa tgatcgtgac attcagcgca tccagtcaga ttttgagagg gcctttgctg 1440

agaaaaacac acaggagctt gttctgttgg ctaaagttat ggctaatgct gctcagcctt 1500

ggggctacaa acctattaca aagctcctac caatccatgg cacagctggt gagcaactgt 1560

cccaatcagt tcacattgaa gccatcctgg ccctgagagg cattgccaaa cagaagccca 1620

aagaggttca gaacttggct ctgcagctgt ttatggacac aactctgcag cctgagcagc 1680

gtatgcttgc cgtcatgaca ctctttgaga ccaacccttc aatggccgtc atgactaatg 1740

ttatcaacgt tgtcaagtct gacaacagcc agccagtgat cagctttact tactctctca 1800

tcaagtctca gtccagaagc acagctaacc cctcagtggc acctgaggct aatattgctc 1860

tcagactctt gggccaaagg agacagagca tgaagctgag caaggctttc aaagcggact 1920

tctacagcca tcctctgatg cttggtgctg ctgcaagtat ttattacatc aatgaggctg 1980

ctaccatcct ccccaaagcg gttatagcta agacgagtgc ctatgtcgct ggagctgctg 2040

ctgatgtttt tgagattgga gtcagaagtg agggattcca ggagtacttc ctgaaaaagg 2100

agagctctga tgtctctgat agaaccacca agatgcagcg catcattaaa gctttcacca 2160

actggaagtc tttgccaatc agcaaattgc tgggctctgt gaatgtcaag gttctgggac 2220

aggaaatcgc ttttgttgac attgacgagc agctcattga ggaggcaatg aggattcgct 2280

ctgaaattga catcaaggag tatggcttaa acttactccg tcacttgttc caaaatggtg 2340

tctccgcaca cttggtcaaa gcagtgatgc ccgctgagat cagacgtgtc atgcctactg 2400

ccgcaggcct gccaatggag cttgccttct acactgttgc tgtcactgca gcaaatgtcc 2460

aggccagatt ccaggccaac cttccacaga acttccatgt ttctgacctt ctgaagcaga 2520

agaccaacat tgaggctgac ataaagccaa gcatggccct caacacattt gctgtgatgg 2580

gaatgaacac tgacatcgtt caggctgcca tagtctcaag agccaaggtc caggtcaacg 2640

tgcccgccaa gatagctgct tctttggact tagctgagaa caactttaag atcagtgctc 2700

ttccggttcg cctctctgaa aatgttgcag ttgctgttga cgttgatact ctggccattg 2760

caagacaggc caaacgggta acccctctga ttcccgaaga tgcttctccc caagcatctt 2820

ccgaaacatc ttcatctgct agcgcctcca attctaggga gatactgggt aacatgcagc 2880

aagttcagga caggcccctt cccacgacaa gagttcccag atctgacaag aagttctgca 2940

ctgttactgc tggagtgaag atctgcatca acatcagctc cagcaatgcc aagttcatca 3000

cagattccgc tctctccaga ctggctggaa agcacgctgt tctcatgtct gtcgaacaat 3060

ctgaaagtga caatgtcgag aagtgggaaa tggagcttca gcttggagcc aaggccgcaa 3120

acaagctgat caaaaacatc aacttggaca tggatgaggt cctagagggc acacctattc 3180

tgtccaaact caagagaatc ctgaatccaa gcatgaaaaa caacacctcc tctagctcct 3240

ccagcagctc caggtccaga gttcgtagca gccatccttc ctcctcatcc tcctcctcct 3300

cctcatcgtc caagtctcac atggccggta aagttatcag caccatgggc aaaatcatcg 3360

gggttaacca aaagaggagc agcagcagca gcagcagcag taggagtcag caaaaccgca 3420

agaggcaaag gtccactgtg tccagcctga gctctctgtt cagtgctagc tccagctcct 3480

cacagttttt ccccaagtct cagcgccaga gctctcgttc caaattccag ccaaaccacc 3540

agaagacgac atccaagcgg cactcaggat ctgcctcctc tgcaagaacc tttgaggaca 3600

tcaggaaaca gaacaaattc cttggcaata ccgttgagcc agtttttgca ctgatcctcc 3660

gtgctgtcag agctaacaac aacaatccac tgggctacca gatcgctgcc tataaggatg 3720

gagacagagt tcagatgatc atggctgccc tggcttctca tgacaactgg aggctttgtg 3780

ctgatgccat taaacttagc aagaacaaag ctgctgctaa aattgcatgg ggagagaagt 3840

gccagaaata tgagaccatg attacagctg agtctggccg tgttcagaac aagggacaat 3900

ctcaagatgc agctcgtgtg agagtggcct ggaaaagact gcccactgct gttgtcaaag 3960

atgtcaaaat gatctacaac tccatcattg ccccttactt gtctagtggc tatctgcaga 4020

agagatcaga tgcgaccagg cagatttcct tcactgtggt tgttgagcct aagaaacagc 4080

ttggctttat ttggaaatca ccagcatttg tctacaggag taatgtgcct cttcccatca 4140

ctctgccaat cgatgagctg aaggaagtgc tgccatttga tgaaatgctt gacaacgctc 4200

actatctgtt ggcaaagact actggaattc agtgcagatt taatgaagga cagcttacca 4260

cttccagcaa aagacaacac aagaactaca tgccaaattc ttgctaccag ctgctggctc 4320

aggattgcac cgatgagctt aaattcattg ttttgctgaa gaaggatagc gcaggccgtt 4380

acatggtcaa tgtgaagatt ggtacaaggg atattgacat gttctttaat ggagaaagac 4440

cagctgtcat gatcaatgga aaggaaattg ccagagagcg cttgccatat gacagagatt 4500

cagtgacgat tctgctgatg aacggcaagc tgtttctccg tgctctggac tttggcattg 4560

ctgaactcca attcagtgca tcagaagtga cgatcaatgt tccagagtat ttgaggaaca 4620

aagtctgcgg tctctgcggc caaggcaatg gagacaggag aaacgattac cgcatgccca 4680

atggacgtat cactgataac cccatcagct tcgcccattc ctggactctg ccctctcaga 4740

gttgcagcga tgagactgaa tgtcgtttga ctcatgagtc cattgaactg gagagagaaa 4800

ttaacgatca cggtgtgccg tctaagtgct tctctgtgga ctctgtgctg cgctgtcgcc4860

ctggctgcac tcccactaag accaccatgt ccagtgtgag cttccactgc agacccctca 4920

atgacaacag ccaagtgtca gatatccgta accgcagcat tgacatgact gagtccgttg 4980

aagcccatct tgactgcagt tgcacttctc agtgtgctta gatcttgtcg tcttatattg 5040

tgtctatgtg taattttaac taaataaatg aaggcatctc aaaagacaaa aaaaaaaaaa 5100

aaaaaaaaaa aaaaaaaaaa tataaagaaa aaaaaaaaaa aaaaaaggta ctctgcgctg 5160

ataccactgc ttgccctata gtgagtcgta ttagaatcga attcccgcgg ccgcatggcg 5220

gcc 5223

<210>2

<211>1665

<212>PRT

<213>Periophthalmus modestus（VtgAa）

<400>2

Met Arg Val Val Val Leu Ala Leu Ala Leu Ala Leu Val Ala Gly Gln

1 5 10 15

His Asp Asn Leu Ala Pro Ser Phe Ala Pro Gly Leu Thr Tyr Val Tyr

20 25 30

Lys Tyr Asn Ala Gln Ser Leu Gly Gly Leu Ser Glu Gln His Leu Ala

35 40 45

Lys Ala Gly Leu Asn Phe Thr Ser Asn Val Lys Ile Ser Val Ala Gln

50 55 60

Glu Asn Val Leu Met Leu Gln Leu Glu Asn Pro Arg Ile Tyr Glu Phe

65 70 7580

Ser Gly Val Trp Pro Lys Asp Ser Tyr Val Gln Thr His Leu Arg Ala

85 90 95

Asp Leu Glu Ala His Leu Lys Thr Ala Ile Lys Phe Lys Tyr Asp His

100 105 110

Gly Ile Val Arg Glu Ile Leu Ala Pro Glu Ser Val Pro Ile Leu Leu

115 120 125

Leu Asn Ile Phe Arg Gly Ile Leu Asn Phe Phe Gln Leu Asn Ile Lys

130 135 140

Lys Ser Gln Asn Val Tyr Glu Leu Gln Glu Glu Gly Ala Gln Gly Val

145 150 155 160

Cys Lys Thr Gln Tyr Ala Ile Thr Glu Asn Asp Lys Ala Glu Arg Ile

165 170 175

Leu Leu Thr Lys Ser Arg Asn Leu Asn His Cys Gln Glu Lys Val Met

180 185 190

Arg Asp Ile Gly Leu Ala Tyr Thr Lys Thr Trp His Lys Cys Arg Glu

195 200 205

Ile Ser Lys Asn Leu Arg Gly Thr Thr Gly Tyr Phe Tyr Lys Leu Lys

210 215 220

Ala Ala Pro Gly Gly Leu Ile Ile Glu Lys Ala Ser Gly Lys Glu Val

225 230 235240

Ile Gln Phe Thr Pro Phe Asn Asp Gln Asn Gly Ala Ala Ser Met Gln

245 250 255

Thr Ile Gln Thr Leu Asp Phe Ile Ala Ala Ile Lys Ala Pro Ile Val

260 265 270

Pro Ile Ser Ala Pro Tyr His Pro Arg Gly Ser Leu Lys Tyr Gln Phe

275 280 285

Ser Thr Glu Leu Leu Gln Ser Pro Leu Arg Ile Leu Lys Met Ser Asp

290 295 300

Val Lys Glu Gln Val Ala Asp Val Leu Asn Asn Leu Val Val Asn Asn

305 310 315 320

Arg Asp Lys Val His Glu Asp Ala Pro Leu Lys Phe Phe Glu Leu Ile

325 330 335

Leu Leu Leu Arg Ala Ser Asp Leu Thr Glu Leu Arg Asn Leu Leu Thr

340 345 350

Thr Tyr Lys Ser Arg Pro Leu Glu Arg Arg Trp Leu Met Asp Ala Ile

355 360 365

Ser Asn Thr Gly Thr Lys Ala Ala Leu Glu Ile Val Met Ala Glu Ile

370 375 380

Gln Lys Arg Glu Leu Ser Val Pro Glu Ala Ala Gln Val Leu Ile Gly

385 390 395 400

Thr Leu His Met Leu Lys Pro Thr Asp Glu Ile Ile Gln Lys Val Trp

405 410 415

Ser Tyr Ile Glu Gln Leu Ser Gln Glu Gln Gly Arg Tyr Glu Gln Val

420 425 430

Val Arg Lys Ala Leu Phe Leu Gly Tyr Gly Ser Ile Ile His Arg Gln

435 440 445

Ser Val Gln Arg Ala Glu Trp Asn Asp Arg Asp Ile Gln Arg Ile Gln

450 455 460

Ser Asp Phe Glu Arg Ala Phe Ala Glu Lys Asn Thr Gln Glu Leu Val

465 470 475 480

Leu Leu Ala Lys Val Met Ala Asn Ala Ala Gln Pro Trp Gly Tyr Lys

485 490 495

Pro Ile Thr Lys Leu Leu Pro Ile His Gly Thr Ala Gly Glu Gln Leu

500 505 510

Ser Gln Ser Val His Ile Glu Ala Ile Leu Ala Leu Arg Gly Ile Ala

515 520 525

Lys Gln Lys Pro Lys Glu Val Gln Asn Leu Ala Leu Gln Leu Phe Met

530 535 540

Asp Thr Thr Leu Gln Pro Glu Gln Arg Met Leu Ala Val Met Thr Leu

545 550 555 560

Phe Glu Thr Asn Pro Ser Met Ala Val Met Thr Asn Val Ile Asn Val

565 570 575

Val Lys Ser Asp Asn Ser Gln Pro Val Ile Ser Phe Thr Tyr Ser Leu

580 585 590

Ile Lys Ser Gln Ser Arg Ser Thr Ala Asn Pro Ser Val Ala Pro Glu

595 600 605

Ala Asn Ile Ala Leu Arg Leu Leu Gly Gln Arg Arg Gln Ser Met Lys

610 615 620

Leu Ser Lys Ala Phe Lys Ala Asp Phe Tyr Ser His Pro Leu Met Leu

625 630 635 640

Gly Ala Ala Ala Ser Ile Tyr Tyr Ile Asn Glu Ala Ala Thr Ile Leu

645 650 655

Pro Lys Ala Val Ile Ala Lys Thr Ser Ala Tyr Val Ala Gly Ala Ala

660 665 670

Ala Asp Val Phe Glu Ile Gly Val Arg Ser Glu Gly Phe Gln Glu Tyr

675 680 685

Phe Leu Lys Lys Glu Ser Ser Asp Val Ser Asp Arg Thr Thr Lys Met

690 695 700

Gln Arg Ile Ile Lys Ala Phe Thr Asn Trp Lys Ser Leu Pro Ile Ser

705 710 715 720

Lys Leu Leu Gly Ser Val Asn Val Lys Val Leu Gly Gln Glu Ile Ala

725 730 735

Phe Val Asp Ile Asp Glu Gln Leu Ile Glu Glu Ala Met Arg Ile Arg

740 745 750

Ser Glu Ile Asp Ile Lys Glu Tyr Gly Leu Asn Leu Leu Arg His Leu

755 760 765

Phe Gln Asn Gly Val Ser Ala His Leu Val Lys Ala Val Met Pro Ala

770 775 780

Glu Ile Arg Arg Val Met Pro Thr Ala Ala Gly Leu Pro Met Glu Leu

785 790 795 800

Ala Phe Tyr Thr Val Ala Val Thr Ala Ala Asn Val Gln Ala Arg Phe

805 810 815

Gln Ala Asn Leu Pro Gln Asn Phe His Val Ser Asp Leu Leu Lys Gln

820 825 830

Lys Thr Asn Ile Glu Ala Asp Ile Lys Pro Ser Met Ala Leu Asn Thr

835 840 845

Phe Ala Val Met Gly Met Asn Thr Asp Ile Val Gln Ala Ala Ile Val

850 855 860

Ser Arg Ala Lys Val Gln Val Asn Val Pro Ala Lys Ile Ala Ala Ser

865 870 875 880

Leu Asp Leu Ala Glu Asn Asn Phe Lys Ile Ser Ala Leu Pro Val Arg

885 890 895

Leu Ser Glu Asn Val Ala Val Ala Val Asp Val Asp Thr Leu Ala Ile

900 905 910

Ala Arg Gln Ala Lys Arg Val Thr Pro Leu Ile Pro Glu Asp Ala Ser

915 920 925

Pro Gln Ala Ser Ser Glu Thr Ser Ser Ser Ala Ser Ala Ser Asn Ser

930 935 940

Arg Glu Ile Leu Gly Asn Met Gln Gln Val Gln Asp Arg Pro Leu Pro

945 950 955 960

Thr Thr Arg Val Pro Arg Ser Asp Lys Lys Phe Cys Thr Val Thr Ala

965 970 975

Gly Val Lys Ile Cys Ile Asn Ile Ser Ser Ser Asn Ala Lys Phe Ile

980 985 990

Thr Asp Ser Ala Leu Ser Arg Leu Ala Gly Lys His Ala Val Leu Met

995 1000 1005

Ser Val Glu Gln Ser Glu Ser Asp Asn Val Glu Lys Trp Glu Met

1010 1015 1020

Glu Leu Gln Leu Gly Ala Lys Ala Ala Asn Lys Leu Ile Lys Asn

1025 1030 1035

Ile AsnLeu Asp Met Asp Glu Val Leu Glu Gly Thr Pro Ile Leu

1040 1045 1050

Ser Lys Leu Lys Arg Ile Leu Asn Pro Ser Met Lys Asn Asn Thr

1055 1060 1065

Ser Ser Ser Ser Ser Ser Ser Ser Arg Ser Arg Val Arg Ser Ser

1070 1075 1080

His Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser

1085 1090 1095

His Met Ala Gly Lys Val Ile Ser Thr Met Gly Lys Ile Ile Gly

1100 1105 1110

Val Asn Gln Lys Arg Ser Ser Ser Ser Ser Ser Ser Ser Arg Ser

1115 1120 1125

Gln Gln Asn Arg Lys Arg Gln Arg Ser Thr Val Ser Ser Leu Ser

1130 1135 1140

Ser Leu Phe Ser Ala Ser Ser Ser Ser Ser Gln Phe Phe Pro Lys

1145 1150 1155

Ser Gln Arg Gln Ser Ser Arg Ser Lys Phe Gln Pro Asn His Gln

1160 1165 1170

Lys Thr Thr Ser Lys Arg His Ser Gly Ser Ala Ser Ser Ala Arg

1175 1180 1185

Thr Phe Glu Asp Ile Arg Lys Gln Asn Lys Phe Leu Gly Asn Thr

11901195 1200

Val Glu Pro Val Phe Ala Leu Ile Leu Arg Ala Val Arg Ala Asn

1205 1210 1215

Asn Asn Asn Pro Leu Gly Tyr Gln Ile Ala Ala Tyr Lys Asp Gly

1220 1225 1230

Asp Arg Val Gln Met Ile Met Ala Ala Leu Ala Ser His Asp Asn

1235 1240 1245

Trp Arg Leu Cys Ala Asp Ala Ile Lys Leu Ser Lys Asn Lys Ala

1250 1255 1260

Ala Ala Lys Ile Ala Trp Gly Glu Lys Cys Gln Lys Tyr Glu Thr

1265 1270 1275

Met Ile Thr Ala Glu Ser Gly Arg Val Gln Asn Lys Gly Gln Ser

1280 1285 1290

Gln Asp Ala Ala Arg Val Arg Val Ala Trp Lys Arg Leu Pro Thr

1295 1300 1305

Ala Val Val Lys Asp Val Lys Met Ile Tyr Asn Ser Ile Ile Ala

1310 1315 1320

Pro Tyr Leu Ser Ser Gly Tyr Leu Gln Lys Arg Ser Asp Ala Thr

1325 1330 1335

Arg Gln Ile Ser Phe Thr Val Val Val Glu Pro Lys Lys Gln Leu

1340 1345 1350

Gly Phe Ile Trp Lys Ser Pro Ala Phe Val Tyr Arg Ser Asn Val

1355 1360 1365

Pro Leu Pro Ile Thr Leu Pro Ile Asp Glu Leu Lys Glu Val Leu

1370 1375 1380

Pro Phe Asp Glu Met Leu Asp Asn Ala His Tyr Leu Leu Ala Lys

1385 1390 1395

Thr Thr Gly Ile Gln Cys Arg Phe Asn Glu Gly Gln Leu Thr Thr

1400 1405 1410

Ser Ser Lys Arg Gln His Lys Asn Tyr Met Pro Asn Ser Cys Tyr

1415 1420 1425

Gln Leu Leu Ala Gln Asp Cys Thr Asp Glu Leu Lys Phe Ile Val

1430 1435 1440

Leu Leu Lys Lys Asp Ser Ala Gly Arg Tyr Met Val Asn Val Lys

1445 1450 1455

Ile Gly Thr Arg Asp Ile Asp Met Phe Phe Asn Gly Glu Arg Pro

1460 1465 1470

Ala Val Met Ile Asn Gly Lys Glu Ile Ala Arg Glu Arg Leu Pro

1475 1480 1485

Tyr Asp Arg Asp Ser Val Thr Ile Leu Leu Met Asn Gly Lys Leu

1490 1495 1500

Phe Leu Arg Ala Leu Asp Phe Gly Ile Ala Glu Leu Gln Phe Ser

1505 1510 1515

Ala Ser Glu Val Thr Ile Asn Val Pro Glu Tyr Leu Arg Asn Lys

1520 1525 1530

Val Cys Gly Leu Cys Gly Gln Gly Asn Gly Asp Arg Arg Asn Asp

1535 1540 1545

Tyr Arg Met Pro Asn Gly Arg Ile Thr Asp Asn Pro Ile Ser Phe

1550 1555 1560

Ala His Ser Trp Thr Leu Pro Ser Gln Ser Cys Ser Asp Glu Thr

1565 1570 1575

Glu Cys Arg Leu Thr His Glu Ser Ile Glu Leu Glu Arg Glu Ile

1580 1585 1590

Asn Asp His Gly Val Pro Ser Lys Cys Phe Ser Val Asp Ser Val

1595 1600 1605

Leu Arg Cys Arg Pro Gly Cys Thr Pro Thr Lys Thr Thr Met Ser

1610 1615 1620

Ser Val Ser Phe His Cys Arg Pro Leu Asn Asp Asn Ser Gln Val

1625 1630 1635

Ser Asp Ile Arg Asn Arg Ser Ile Asp Met Thr Glu Ser Val Glu

1640 1645 1650

Ala His Leu Asp Cys Ser Cys Thr Ser Gln Cys Ala

1655 1660 1665

<210>3

<211>26

<212>PRT

<213>Artificial Sequence

<220>

<223>vtgAa-degenerate-F1

<400>3

Ala Ala Arg Ala Thr Gly Ala Ala Gly Cys Gly Tyr Arg Thr Cys Met

1 5 10 15

Thr Asp Met Ala Gly Gly Cys Thr Cys Thr

20 25

<210>4

<211>26

<212>PRT

<213>Artificial Sequence

<220>

<223>vtgAa-degenerate-R1

<400>4

Ala Ala Cys Trp Gly Gly Met Ala Gly Arg Arg Cys Thr Tyr Ser Asp

1 5 10 15

Ala Lys Cys Thr Thr Ala Ala Ala Ser Thr

20 25

<210>5

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α-F1

<400>5

gccctgctgg cctacaccct 20

<210>6

<211>21

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α-R1

<400>6

ggtggttcag gatgatgacc t 21

<210>7

<211>27

<212>DNA

<213>Artificial Sequence

<220>

<223>vtgAa-5'RACE-GSP1

<400>7

cctggacctt ggctcttgag actatgg 27

<210>8

<211>28

<212>DNA

<213>Artificial Sequence

<220>

<223>vtgAa-3'RACE-GSP2

<400>8

gtcacttgtt ccaaaatggt gtctccgc 28

<210>9

<211>23

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α-5'RACE –GSP1

<400>9

cgtttccacg acggatttcc ttg 23

<210>10

<211>23

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α-3'RACE -GSP2

<400>10

tgtcctggtt caagggatgg aag 23

<210>11

<211>27

<212>DNA

<213>Artificial Sequence

<220>

<223>vtgAa-full primer-F1

<400>11

cagccatgag agtcgttgta ctagcct 27

<210>12

<211>25

<212>DNA

<213>Artificial Sequence

<220>

<223>vtgAa-full primer-R1

<400>12

caagcagtgg tatcagcgca gagta 25

<210>13

<211>23

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α-full primer-F1

<400>13

gccttgaaaa acccagaaac accg 23

<210>14

<211>25

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α-full primer-R1

<400>14

ggtgatgtga ggggaaaggg ag 22

<210>15

<211>

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α qPCR-F1

<400>15

tggtgacagc aagaacaacc 20

<210>16

<211>

<212>DNA

<213>Artificial Sequence

<220>

<223>ef1-α qPCR-R1

<400>16

atgaacttgc aggcgatgtg 20

<210>17

<211>

<212>DNA

<213>Artificial Sequence

<220>

<223>vtgAa-qPCR-F1

<400>17

aaattgccag agagcgcttg 20

<210>18

<211>

<212>DNA

<213>Artificial Sequence

<220>

<223>vtgAa-qPCR-R1

<400>18

aacagcttgc cgttcatcag 20

Claims

1. The application of the mackerel vitellogenin as an indicator protein for detecting environmental estrogen pollution is disclosed, wherein the amino acid sequence of the mackerel vitellogenin is shown as SEQ ID NO. 2.

2. The use according to claim 1, wherein the nucleotide sequence of the mudskipper VtgAa gene encoding the vitellogenin is as shown in SEQ ID No.1 from position 3 to position 5000.

3. A test method for detecting aquatic environment pollution, which is characterized by comprising the step of detecting the expression level of mackerel vitellogenin in fish in an aquatic environment, wherein the amino acid sequence of the mackerel vitellogenin is shown as SEQ ID No. 2.

4. A test method for detecting aquatic environmental pollution according to claim 3, wherein the aquatic environmental pollution is an estrogenic effect.

5. A test method for detecting aquatic environmental pollution according to claim 3, wherein said method includes the steps of:

s2, designing VtgAa and internal reference ef 1-alpha gene fluorescent quantitative PCR primers respectively, and establishing an absolute quantitative PCR method; wherein, the fluorescent quantitative PCR primer of the VtgAa gene is vtgAa-qPCR-F1: 5'-AAATTGCCAGAGAGCGCTTG-3' and vtgAa-qPCR-R1: 5'-AACAGCTTGCCGTTCATCAG-3', respectively; the fluorescent quantitative PCR primer of the internal reference ef 1-alpha gene is ef 1-alpha qPCR-F1: 5'-TGGTGACAGCAAGAACAACC-3' and ef1- α qPCR-R1: 5'-ATGAACTTGCAGGCGATGTG-3', respectively;

6. A method of cloning the full-length sequence of the mudskipper VtgAa gene, the method comprising the steps of:

a4, designing a specific full-length primer, and amplifying to obtain a VtgAa gene full-length sequence;

in the step A2, the degenerate primer corresponding to the VtgAa gene is vtgAa-degenerate-F1: 5 '-AARATGAAGCGYRTCMTDMAGGCTCT-3' and vtgAa-degenerate-R1: 5 '-AACWGGMAGRRCTYSDAKCTTAAAST-3';

in the step A3, the RACE primers corresponding to the VtgAa gene are vtgAa-5'RACE-GSP1 shown in SEQ ID NO.7 and vtgAa-3' RACE-GSP2 shown in SEQ ID NO. 8;

in step A4, the specific full-length primers corresponding to the VtgAa gene are vtgAa-fullprimer-F1 shown in SEQ ID NO.11 and vtgAa-full primer-R1 shown in SEQ ID NO. 12.