CN106831973A

CN106831973A - Water body estrogen contamination detection method based on many types of vitellogenin of mudskipper

Info

Publication number: CN106831973A
Application number: CN201710163953.3A
Authority: CN
Inventors: 吴美琴; 陈渊戈; 葛珂珂; 练青平
Original assignee: Ninghai County Sanmenwan Modern Fisheries Park Management Committee; Shanghai Maritime University; East China Sea Fishery Research Institute Chinese Academy of Fishery Sciences; Zhejiang Institute of Freshwater Fisheries
Current assignee: Ninghai County Sanmenwan Modern Fisheries Park Management Committee; Shanghai Maritime University; 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: 2017-06-13
Anticipated expiration: 2037-03-17
Also published as: CN106831973B

Abstract

The invention discloses a kind of water body estrogen contamination detection method based on mudskipper vitellogenin, belong to molecular biology and environmental pollution detection field；The amino acid sequence of the mudskipper vitellogenin is as shown in SEQ ID N0.2.Not yet there are beach fish to be detected for the biological living of environmental estrogens at present, its corresponding Measurement for Biotechnique lacks.The strong mudskipper Periophthalmus modestus living sample sensitive to habitat of present invention selection beach colonization property, design primer, the expression of its vitellogenin VtgAa is detected using real-time quantitative fluorescence PCR method, the detection method can be used for the detection of the water environment estrogen pollution level such as seawater, mouth of the river.

Description

Water body estrogen contamination detection method based on many types of vitellogenin of mudskipper

Technical field

The invention belongs to molecular biology and environmental pollution detection field, and in particular to one kind is based on new beach pattern fish The water body environment estrogen detection method of many types of vitellogenin of class-mudskipper (vitellogenin, vtg), can extensive utilization In the assessment of the environmental estrogens pollution level in river mouth beach waters.

Background technology

In the present age of industry high speed development, a large amount of chemical substances unreasonably discharge in the environment, modern agriculture is to agricultural chemicals ground Undue dependence all causes very big ground contamination to environment.Increasing evidence shows there is various can simulation and dry in environment The material of disturbance thing and human endocrinological's function.After these exogenous compounds enter body, the endocrine synthesis of interference body, Release, transport, metabolism, with reference to etc. process, and thereby result in the lesions such as change and the deformity of the male and female sex of animal.This is big It is female sharp that class material is referred to as environment incretion interferent (Environmental Endocrine Disrupters, EEDs), environment Plain (Enviromental estrogens) or endocrine activity compound (Endocrine active compounds).Ring Border estrogen problem is mentioned in the same breath with depletion of the ozone layer and greenhouse effects, is reflected to its attention degree.

The research work of major part incretion interferent at present is also directed to the simulation exposure of single compound, actual environment System but includes a series of materials with different estrogen actives.So, it is right that the toxicology data based on single substance is difficult Ecological risk is accurately assessed.The estrogen active of research mixture more meets actual environment and is deposited than research single substance State.The mixture being made up of several estrogen-like matters of invisible effective concentration has obvious estrogen active. Economic, convenience, sensitive environmental estrogens mixture activity test method are set up, particularly finds efficient, special, sensitive raw Thing mark has turned into the focus of environmental endocrine disruptors research.

Environmental estrogens species is various, and chemical analysis can not make a thorough inquiry about all dirts with estrogen effect in environmental sample Dye thing, and the power of its poisonous effect only can not be learnt by the concentration of environmental estrogens.Again, chemical analysis method is to instrument The dependency degree of device is high, takes time and effort.And the utilization of biomarker solves this defect well, by the whole of determination sample Body endocrine disrupting and the detailed compound group of sample need not be known into and simple to operate, quick, economical and efficient excellent Point, has become the Main Means of monitoring of environmental incretion interferent.

Mudskipper Periophthalmus modestus, are under the jurisdiction of Perciformes, goby suborder, mudskipper section, mudskipper Category, inhabit river mouth salt-fresh water waters, at offshore beach or substrate mud low tidal region, be the wide temperature eurysalinity fish of littoral warm water Class.

The vitellogenin (vitellogenin, Vtg) of fish is under estradiol (estradiol-17 β, E2) effect The yolk forerunner's albumen in blood is secreted into after synthesizing through liver.Under normal circumstances, Vtg is raun in yolk shaping age blood The female specific serum albumen of middle appearance, but milter and the postlarva Vtg in same energy inductor under exogenous estrogen action Generation.According to this characteristic, Vtg, also can be by detection in addition to can be as the indicator protein for judging sex and raun maturity The abnormal of Vtg generates to evaluate the pollution of water environment exogenous estrogen substance (environmental estrogens) in milter or postlarva body Situation (Specker＆Sullivan, 1994).Oestrogen-like hormone dirt is developed in existing part for vitellogenin both at home and abroad at present Dye detecting method, but selected fish models are mainly fresh water cyprinid fish, have no that beach waters fish swash for environment to be female The patent report of plain context of detection.And fresh water cyprinid fish and the water body of mudskipper life are entirely different, mudskipper is given birth to due to it The particularity in border, the application in environmental estrogens detection has the irreplaceable practical significance of cyprinid fish.This research is selected Beach colonization property mudskipper establishes the live body environment measuring technology for vitellogenin, with higher as living sample Innovation and practicality.

The content of the invention

It is an object of the invention to provide a kind of water body estrogen pollution detection based on many types of vitellogenin of mudskipper Method, is applied to the environmental estrogens pollution detection in each waters.

The purpose of the present invention is achieved through the following technical solutions：

In a first aspect, the present invention relates to a kind of mudskipper vitellogenin in the instruction polluted as detection environmental estrogens Purposes in albumen, the amino acid sequence of the mudskipper vitellogenin is as shown in SEQ ID NO.2.

Preferably, the nucleotide sequence such as SEQ ID of the mudskipper VtgAa genes of the mudskipper vitellogenin are encoded In NO.1 shown in the 3rd~the 5000th.

Second aspect, the present invention relates to a kind of method of testing for detecting aquatic environment pollution, methods described includes detection water Fish in raw environment are hit by a bullet and apply the expression of fish vitellogenin, and the amino acid sequence of the mudskipper vitellogenin is such as Shown in SEQ ID NO.2.

Preferably, the aquatic environment pollution is estrogen effect.

Preferably, methods described comprises the following steps：

S1, from the liver organization of mudskipper juvenile fish or milter total serum IgE is extracted, carry out post transcription cloning；

S2, VtgAa and internal reference ef1- α gene by fluorescence quantitative PCR primers are separately designed, set up absolute quantitation PCR methods；

S3, the mRNA expressions to the vitellogenin VtgAa in sample are quantified, and the environment for analyzing sample is female sharp Plain pollution level.

The third aspect, the present invention relates to a kind of method for cloning mudskipper VtgAa full length gene sequences, methods described includes Following steps：

A1, from the liver organization of sexual maturing period mudskipper raun total serum IgE is extracted, carry out post transcription cloning；

A2, the degenerate primer of design VtgAa carry out amplified reaction, obtain VtgAa genetic fragments；

A3, with mudskipper total serum IgE as template according to RACE methods prepare respectively VtgAa genes 3 ' end and 5 ' end cDNA sequences；

The specific total length primer of A4, design, amplification obtains VtgAa full length gene sequences.

Preferably, in step A2, the corresponding degenerate primer of VtgAa genes is the vtgAa- as shown in SEQ ID NO.3 The degenerate-F1 and vtgAa-degenerate-R1 as shown in SEQ ID NO.4.

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

Preferably, in step A4, the corresponding specific total length primer of VtgAa genes is as shown in SEQ ID NO.11 The vtgAa-full primer-F1 and vtgAa-full primer-R1 as shown in SEQ ID NO.12.

Compared with prior art, the present invention has the advantages that：

1st, present invention obtains mudskipper VtgAa full length gene CDS sequences, the sky of mudskipper Vtg gene families has been filled up In vain.

2nd, mudskipper is the widely distributed common river mouth fish of China's southeastern coast, and Vtg is to understand river mouth salt-fresh water water At domain, offshore beach or substrate mud low tidal region environmental estrogens pollution important biomolecule mark, obtain VtgAa genes it is complete It is long, for more accurately indicating corresponding water pollution situation, and deeper into probing into protein expression of the aquatile in polluted water region The problems such as it is significant.

3rd, the present invention establishes the real-time glimmering of mudskipper VtgAa and ef1- α (internal reference) gene by designing specific primer Fluorescent Quantitative PCR method.The situation of change of expressions of the VtgAa under estrogen induction is have detected with fluorescence quantitative PCR method, And then indicate the estrogen pollution condition in environment.

Brief description of the drawings

Fig. 1 is the cDNA total length electrophoretograms of mudskipper vitellogenin VtgAa and ef1- α genes；Wherein, M：Marker, 1：VtgAa, 2：ef1-α；

Fig. 2 is the canonical plotting of the quantitative fluorescent PCR of mudskipper VtgAa genes and reference gene ef1- α；

Fig. 3 is the change of VtgAa expressions under various concentrations estrogen different time is induced.

Specific embodiment

With reference to embodiment, the present invention is described in detail.Following examples will be helpful to those skilled in the art The present invention is further understood, but the invention is not limited in any way.It should be pointed out that to one of ordinary skill in the art For, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These belong to guarantor of the invention Shield scope.

Embodiment

The present embodiment be related to a kind of mudskipper many types of vitellogenin (vitellogenin, vtg) gene, encoding proteins and Its application, the method for clone's cDNA full length sequences.

VtgAa genes：The many types of VtgAa genes of mudskipper of the invention, it is characterised in that VtgAa sequence 5223bp, Comprising initiation codon ATG, terminator codon TAG and polyA.The ORFs (ORF) of the gene is located at 3~5000bp, 4998bp long, encodes 1665 amino acid altogether, and wherein signal peptide is 15 amino acid.According to reckoning, its encoding proteins molecular weight It is 185kDa.The encoding amino acid sequence has typical perfect form livetin original structure：1 vitellogenin domain (being located at 24~No. 594 amino acid of coded amino acid peptide chain), 1 VWFD domain (are located at the 1423 of coded amino acid peptide chain ~No. 1555 amino acid), 1 Poly(Ser) region (being located at 1064~No. 1195 amino acid of coded amino acid peptide chain), separately It is outer also to contain 1 proteolytic cleavage site (RSRR).By the derivation amino acid sequence and other fish Vtg amino acid sequence ratios To rear discovery, the amino acid sequence includes 4 parts of LvH, LvL, Pv and β component.

The nucleotide sequence of mudskipper VtgAa genes of the present invention is as shown in SEQ ID NO.1.

The code area of the total length VtgAa cDNA sequences for being obtained is from the 3rd nucleotides to 5000 nucleotides.

The amino acid sequence of mudskipper VtgAa genes of the present invention is as shown in SEQ ID NO.2.

By designing specific primer, primer is as shown in SEQ ID NO.15~18, to establish mudskipper to the present invention The real time fluorescence quantifying PCR method of VtgAa and ef1- α (internal reference) gene.VtgAa genes are have detected with fluorescence quantitative PCR method Expression change under the induction of various concentrations estrogen, as a result shows：As the increase inductive effect of concentration is significantly increased；With There is obvious time dependence effect in the increase of open-assembly time, VtgAa expression quantity (same concentration).

The method for cloning cDNA full length sequences：Total serum IgE is extracted from the liver organization of sexual maturing period mudskipper raun, is carried out Post transcription cloning, designs the degenerate primer of Vtg, obtains corresponding gene fragment；On this basis, obtained using SMART RACE technologies Obtain 5 ' and 3 ' end unknown nucleotide sequences；Total length upstream and downstream primer is finally designed, clone obtains corresponding full length cDNA sequence.With Macvector software certifications entire open reading frame (ORF) simultaneously analyze its each functional domain.Compared by complete amino acid sequence and built Erection system analysis chart, determines its Vtg type.

In implementation process, the technical scheme for cloning cDNA full length sequences can use following specific steps：

1st, animal tissue's organ samples collection：

The liver of the fresh bosom ovum mudskipper raun of collection, is placed in RNAlater after shredding and preserves for Total RNAs extraction.

2nd, the extraction of mudskipper total serum IgE and post transcription cloning

Total serum IgE is extracted according to ISOGEN II kits operating instruction, specific method is as follows：

(1) liver organization (about 100mg) is put into 1.5ml centrifuge tubes, adds 1ml ISOGEN II, electronic homogenate rod is even After slurry, 0.4ml RNase free water, 15s is added acutely after vibration, to be stored at room temperature 10min；At 4 DEG C, 12K × g, centrifugation 10min。

(2) supernatant 1ml is taken, adds 5 μ l para-bromoanisole, 15s acutely after vibration, to be stored at room temperature 3min；At 4 DEG C, 12K × g, is centrifuged 10min.

(3) supernatant 1ml is taken, 1ml (isometric) isopropanol is added, 10min is stored at room temperature after reverse mixing；At 4 DEG C, The white precipitate of 12K × g, centrifugation 10min, ttom of pipe and tube wall is RNA precipitate.

(4) supernatant is abandoned, the ethanol of 0.5ml 75% washing precipitation, at 4 DEG C, 7K × g, centrifugation is added in RNA precipitate 3min, abandons supernatant；Repeat this step once.

(5) the drying precipitated 2min of room temperature shading.

(6) 50 μ l (or appropriate) RNase free water dissolves precipitation is added, this solution is total rna solution.

(7) with 0.8% agarose/TAE gel electrophoresis detection total serum IgE integrality；With Thermo Fisher NanoDrop 2000 spectrophotometric determination total rna concentrations and purity.

(8) by above-mentioned total serum IgE according to VILO^TMCDNA Synthesis Kit (Invitrogen) are anti- It is transcribed into cDNA templates.

Note：The above centrifuge tube, homogenate rod etc. are RNase free treatment.

2nd, degenerate primer amplification Vtg genes and ef1- α reference gene fragments

Vtg genes belong to has conservative in sequence, according to halibut, porgy, grey mullet, mosquito fish, lance acanthogobius vtg Gene C DS sequences, sequence alignment is carried out with Blast, and the conserved sequence according to Vtg genes designs a pair of degenerate primers, according to The conserved sequence of ef1- α genes designs a pair of specific primers, and 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

Pcr amplification reaction system is 10 μ l systems：Each 1 μ l, cDNA template 0.5 μ l, ddH of F and R ends primer (10 μM)₂O The μ l of 2.5 μ l, Master Mix 5, reaction condition is 95 DEG C of predegeneration 5min, 35 circulations (95 DEG C, 30s；54 DEG C, 30s；72 DEG C, 1min), 72 DEG C of extension 10min.

4th, the recovery and purifying of PCR primer

Taking 5 μ l PCR reactants carries out 1.5% agarose gel electrophoresis, for confirming intermediate sequence amplified production.Will Target stripe product GENECLEAN Turbo Kit (MP Biomedicals Europe, France) tap rubber to purify and reclaim.

5th, the connection conversion of PCR amplifications purpose fragment

(1) connect：μ l, pGEM-T Easy Vector (Promega, USA) of PCR primer 3 is added in 0.2ml PCR pipes 1 μ l, T4 ligase 1,5 μ l of μ l, Buffer, after gently mixing, 4 DEG C stand overnight.

(2) convert：Plus the μ l of connection product 5 flick mixing, ice bath 30min in bacillus coli DH 5 alpha competent cell；42 DEG C heat shock 45s, is placed in 2min on ice at once；Flat board is applied, culture dish is inverted into 37 DEG C of incubators overnight.

6th, the identification of positive colony, sequencing and sequence analysis

The following mixed liquors of 10ul are added in 0.2ml PCR pipes：AmpliTaqMaster Mix 5 μ l, T7 and SP6 primers (10 μM) each 0.5 μ l, sterilized water 4ul, is put into above-mentioned PCR pipe after taking monospecific polyclonal with sterile toothpick, is allowed to abundant Bacterium colony PCR checkings are carried out after contact.PCR reaction conditions are as follows：95 DEG C of predegeneration 5min；(95 DEG C are denatured 30s, 50 for 35 circulations DEG C annealing 30s, 72 DEG C extension 1min)；72 DEG C of extension 10min.Confirm the clone comprising purpose fragment, Amplification Culture, with T7 and SP6 primers are sequenced.Result obtains 579bp VtgAa and 640bp ef1- alpha gene fragments respectively.

7th, 5 ' RACE and 3 ' RACE amplification mudskippers Vtg5 ' ends and 3 ' end gene orders

It is as follows using Macvector design RACE primers：

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

According toThe Kit of RACE 5 '/3 ' (Clontech) specification is carried out, with mudskipper total serum IgE as template Its 3 ' end and 5 ' end cDNA are prepared, the PCR reaction solutions that cumulative volume is 25 μ l are configured to specific primer (GSP)：Including 2.5 μ L10 × Advantage 2 PCR bufier, 0.5 μ l dNTPs mix (10mM), 0.5 μ l GSP (10 μM), 2.5 μ l 10 × UPM, 1.25 μ l templates and the polymerase mix (Clontech) of 0.5 10 × Advantage of μ l 2.PCR reaction conditions are such as Under：5 circulations (94 DEG C, 30s；72 DEG C, 3min), 5 circulations (94 DEG C, 30s；70 DEG C, 30s；72 DEG C, 3min), 25 circulations (94 DEG C, 30s；68 DEG C, 30s；72 DEG C, 3min).

5 ' RACE and 3 ' RACE connect step of converting with 5 after entering performing PCR reaction.

8th, VtgAa full length gene sequences are expanded

3 ' the ends obtained according to RACE methods and 5 ' end cDNA sequences, separately design specific total length primer as follows：

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

Configure following PCR reaction solutions：Including the PCR buffer of 10 × Advantage 2 (5 μ l), dNTPs mix (10mM, 1 μ l), upstream and downstream primer GSP (10 μM, each 1 μ l), template (1 μ l) and polymerase mix (1 μ of 10 × Advantage 2 L), plus N.F.W. to total reaction volume be 50 μ l.PCR reaction conditions are as follows：95 DEG C of predegeneration 1min；5 circulation (95 DEG C, 30s；68 DEG C, 3min), 25 circulations (94 DEG C, 30s；68 DEG C, 30s；72 DEG C, 3min), 68 DEG C of extension 3min.

1% agarose gel electrophoresis of PCR primer detects expanding effect as shown in figure 1, as shown in Figure 1, with cDNA as mould Plate, obtains the VtgAa bands of expected total length 5000bp or so with the amplification of SEQ ID19 and SEQID20 primer respectively；With SEQ The amplification of ID23 and SEQID24 primers obtains the ef1- α bands of expected total length 1500bp or so.Wherein, M：Marker, 1： VtgAa, 2：ef1-α.

9th, estrogen inducing effect experiment

(1) test fish picks up from the natural pollution-free waters of Shanghai Chongming island Dongtan wetland, and juvenile fish is temporarily supported in 10 10L glass In tank, water temperature 18, pH value 7.5 changes water once daily.

(2) test-induced agent：Endogenous estrogen estradiol (E2)；Cosolvent：Absolute ethyl alcohol.

(3) induction experiment：Implement to lure mudskipper vitellogenin using the method for exposing dissolving estrogen in water body Lead experiment.Experiment be divided into E2 groups, control group, each treatment be divided into 2 it is parallel.E2 groups be provided with 3 exposure concentrations (10ng/L, 100ng/L、1000ng/L).Control group is divided into blank and virgin control, and isodose helping is added in blank control group water body Solvent absolute ethyl alcohol, and then without any reagent in virgin control group.0,2 and 7 days after exposure are entered to each treatment group Row grab sample, every group of 5 tails, live body takes liver organization after anesthesia, after shredding add RNAlater preserve liquid in preserve for Subsequent experimental.

10th, sample treatment

Liver total RNA, spectrophotometer identification RNA mass and detection RNA concentration are extracted using ISOGEN II kits, 1 μ g RNA are taken, using Invitrogen companies reverse transcription reagent box VILO^TM cDNA Synthesis Kit Into cDNA, 10 μ l reaction systems include 2 μ l 5X VILO for reverse transcription^TMReaction Mix, 1 μ l 10X Enzyme Mix, 1 μ g RNA and DEPC-treated water.Response procedures are：25 DEG C, 10min；42 DEG C, 60min；85 DEG C, 5min.Reverse transcription obtains the first chain cDNA, for next step experiment.

11st, quantitative real-time PCR is set up

The recombinant plasmid containing VtgAa and ef1- α (internal reference) genetic fragment is extracted from Escherichia coli respectively, in light splitting light Its DNA concentration and purity are measured on degree meter.With the plasmid cDNA (target gene and reference gene) of preparation for template, it is diluted to 10¹To 10⁶6 gradients of copy number, 2 repetitions of each gradient, the standard for making genes of interest Vtg and ef1- α (internal reference) gene is bent Line (Fig. 2).The actual absolute quantitation of genes of interest is calculated according to standard curve.

Real-time fluorescence quantitative PCR reaction is using Power SYBR Green PCR Master Mix (Applied Biosystems, USA) 20 μ l reaction systems of configuration：2 μ l cDNA, F and R primer (final concentration 200nM), SYBR Green Mix (10 μ l), N.F.W.Carry out simultaneously without control between reverse transcription sample control and group.PCR reaction conditions are：1 is followed Ring (50 DEG C, 2min；95 DEG C, 10min), 40 circulations (95 DEG C, 15s；60 DEG C, 1 min).

According to the vitellogenin VtgAa full length cDNA sequences for having obtained, using Primer3Plus (http：// Www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cg i/) design following primer and carry out reality When quantitative fluorescent PCR reaction.

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

12nd, the estrogen induction result of VtgAa genes

From expression of results of Fig. 3 real-time fluorescence quantitative PCRs detection VtgAa genes under E2 exposures, with ef1- α genes As crt gene, there is expression in exposure group, and in control group without expression.There is expression under low concentration 10ng/L, with The increase inductive effect of concentration is significantly increased；From open-assembly time, to there is the obvious time in each concentration exposure group interdependent again Effect.

SEQUENCE LISTING

<110>Shanghai Ocean University

<120>Water body estrogen contamination 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 cgctgtcgcc 4860

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 75 80

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

225 230 235 240

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 Asn Leu 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

1190 1195 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. as the purposes in detecting the indicator protein that environmental estrogens pollute, the bullet is applied a kind of mudskipper vitellogenin The amino acid sequence of fish vitellogenin is as shown in SEQ ID NO.2.

2. purposes according to claim 1, it is characterised in that the mudskipper of the coding mudskipper vitellogenin The nucleotide sequence of VtgAa genes is as shown in the 3rd~the 5000th in SEQ ID NO.1.

3. it is a kind of to detect the method for testing that aquatic environment pollutes, it is characterised in that methods described is included in detection aquatic environment Fish are hit by a bullet and apply the expression of fish vitellogenin, the amino acid sequence such as SEQ ID of the mudskipper vitellogenin Shown in NO.2.

4. the method for testing that detection aquatic environment according to claim 3 pollutes, it is characterised in that the aquatic environment is dirty Dye is estrogen effect.

5. the method for testing that detection aquatic environment according to claim 3 pollutes, it is characterised in that methods described is included such as Lower step：

S3, the mRNA expressions to the vitellogenin VtgAa in sample are quantified, and the environmental estrogens for analyzing sample are dirty Dye degree.

6. a kind of method for cloning mudskipper VtgAa full length gene sequences, methods described comprises the following steps：

7. the method for cloning mudskipper VtgAa full length gene sequences as claimed in claim 6, it is characterised in that in step A2, The corresponding degenerate primer of VtgAa genes is the vtgAa-degenerate-F1 and such as SEQ ID as shown in SEQ ID NO.3 VtgAa-degenerate-R1 shown in NO.4.

8. the method for cloning mudskipper VtgAa full length gene sequences as claimed in claim 6, it is characterised in that in step A3, The corresponding RACE primers of VtgAa genes are the vtgAa-5 ' RACE-GSP1 and such as SEQ ID NO.8 as shown in SEQ ID NO.7 Shown vtgAa-3 ' RACE-GSP2.

9. the method for cloning mudskipper VtgAa full length gene sequences as claimed in claim 6, it is characterised in that in step A4, The corresponding specific total length primer of VtgAa genes is vtgAa-full primer-F1 as shown in SEQ ID NO.11 and such as VtgAa-full primer-R1 shown in SEQ ID NO.12.