CN1904048A - Zebra fish egg yolk protein origin 1 gene regulating and controlling sequence - Google Patents

Abstract

The present invention belongs to the field of life science. Said invention provides a regulatory controlling sequence expressed by zebra fish vitellogenin 1, (vtg 1) gene of sequence 1. Said invention utilizes the transgenic zebra fish experiment to show that said sequence has promoter activity in the zebra fish body interior, and can make downstream gene implement specific expression in liver under the induction of female sex hormone. By adopting the connection of zfvtg 1-1.7 sequence provided by said invention and reporter gene (fluorescin) the fluorescence can be observed in the living body interior of transgenic zebra fish so as to quickly and visually reflect expression of vtg 1.

Description

Zebra fish vitellogenin-1 gene regulating sequence

Technical field

The invention belongs to life science.Relate to zebra fish vitellogenin-1 (vitellogenin1, vtg1) certain section of genetic expression regulating and controlling sequence.Utilize the transgenic zebrafish experiment, prove that this sequence has promoter activity in the zebra fish body, can under estrogen-induced, start downstream gene the liver specific expression.

Background technology

Vitellogenin (vitellogenin, vtg) be the precursor of three kinds of vitellin(Vt)s, be present in specifically in the ripe oviparity jenny blood, nutrition and functional substance such as amino acid, fat, carbohydrate, VITAMIN, p and s are provided for the embryo who is growing.The vtg of zebra fish is divided into 7 kinds of hypotypes, and vtg1-vtg7 is main only at ripe raun liver expression under the physiological conditions; Be subjected in the water body after the various environmental estrogens effects, milter and juvenile fish can be expressed vtg with growing out of nothing.Wherein the mRNA expression level of vtg1 is than other hypotypes much higher (100-1000 doubly).In recent years, the expression of vtg1 becomes screening and the estrogenic important indicator of testing environment [Marin MG, et al.Mar Pollut Bull.2004 May in the zebra fish body; 48 (9-10): 835-9].

The method that detects zebra fish vtg1 expression level is generally RT-PCR, ELISA, Western-blot.These methods need be extracted RNA or protein, zebra fish must be killed during experiment, adopt expensive reagent (enzyme or antibody), carry out numerous and diverse experiment, the result is static, can not observe directly [Nilsen BM, etal.Anal Bioanal Chem.2004 Feb at live body; 378 (3): 621-33].Maturation gradually along with the zebra fish transgenic technology, make up a kind of transgenic zebrafish of vtg1 expression that can directly reflect and become one of research focus, as: transgenic zebrafish should be able to be expressed certain reporter gene, as green fluorescent protein (enhanced greenfluorescent protein, EGFP), and the expression of the expression of EGFP and vtg1 consistent.By the fluorescent microscope green fluorescence of direct viewing EGFP with the naked eye, just can learn the expression of vtg1, thereby judge whether there is the estrogen activity material in the water body.This transgenic zebrafish have additive method incomparable advantage: can directly observe at live body, method is very easy, does not need the professional, and without any need for reagent, needed only is the logical fluorescent microscope of a Daepori.So inspection cost can be obviously cheap, is easy to popularization and application, especially in many areas that do not have experiment condition, this genetically engineered fish will become people's " environmental protection sentry ".

Make up above-mentioned genetically engineered fish, at first must obtain regulation and control zebra fish vtg1 expression promoter sequence, utilize institute's sequence that obtains to regulate and control the expression of EGFP.CDNA and the protein sequence of zebra fish vtg1 belong to known array, do not appear in the newspapers as yet but regulate and control its expression promoter dna sequence dna.

Summary of the invention

The purpose of this invention is to provide zebra fish vitellogenin-1 (vitellogenin1, vtg1) dna sequence dna of gene regulating sequence, especially zfvtg1-1.7 (zebrafish vitellogenin 1-1.7) with promoter activity.Utilize institute's sequence that obtains to regulate and control the expression of EGFP, reach the estrogenic purpose of testing environment.

The invention provides one section dna sequence dna of model animals spot vitellogenin-1 upstream region of gene of sequence 1, form, called after zfvtg1-1.7 (zebrafishvitellogenin 1-1.7) by 1720 Nucleotide with expression regulation effect.

The present invention adopts cDNA and the protein sequence of known zebra fish vtg1, set up transgenic zebrafish with above-mentioned zfvtg1-1.7 sequence, confirmed that by RT-PCR and hybridization in situ technique zfvtg1-1.7 has following promoter activity in the zebra fish body: 1) be subjected to estrogen-induced; 2) liver specificity.

The present invention at first searches the vtg1 gene in Trust Sanger Institude zebra fish database, and upstream reviews possible promoter sequence, designs primer amplification zebra fish genomic dna, obtains the dna sequence dna of one section 1720bp.

(enhanced greenfluorescent protein EGFP) connects, and obtains fusion gene zfvtg1-1.7-EGFP with the sequence that obtained and enhanced green fluorescence protein gene in the present invention.Described in " zebrafishbook ", zfvtg1-1.7-EGFP is incorporated in the zebra fish genome, make up transgenic zebrafish.With the method for in situ hybridization and RT-PCR, consistent on the time that is expressed in that has proved the expression of transgenic zebrafish EGFP and vtg1 and the space, confirmed the promoter activity of zfvtg1-1.7.

Adopt zfvtg1-1.7 sequence provided by the invention to be connected with reporter gene (fluorescin), can be at the in vivo observation fluorescence of transgenic zebrafish, thus the expression of quicklook ground reflection vtg1.And the expression of vtg1 is the important indicator of reflection estrogen activity, therefore, can judge whether there is estrogen-like product in the water body by the fluorescence of observing the fish body.

The present invention lays a good foundation for making up the fluorescence zebra fish that detects estrogen level, and procreation has positive effect to protection mankind itself safety and health.

Description of drawings

The coexpression of green fluorescent protein and vtg1 (5dpf) in Fig. 1 liver.

Be the zebra fish of 5 days sizes among the figure, the red arrow indication is respectively the expression of green fluorescent protein in the liver (A, B, C) and vtg1 (D, E, F),

Wherein, A, D transgenic zebrafish were cultured 5 days in the system water that contains the female alcohol of 100ng/L 17 alpha-acetylenes,

B, E transgenic zebrafish were cultured 5 days in containing equal-volume solvent-0.001% alcoholic acid system water,

C, F wild-type zebra fish.

Embodiment

Embodiment 1 obtains the dna sequence dna of zfvtg1-1.7

1. the breed of zebra fish

Wild-type zebra fish (Danio rerio) is available from international zebra fish resource center, and the zebra fish cultivating system is introduced from U.S. Aquatic Habitats company, and the nursing scheme is described according to Zebrafish Book and carried out.

2. extract the zebra fish genomic dna

Adopt the genome DNA extracting reagent kit of U.S. promega company to extract the zebra fish genomic dna.

3. design of primers

According to the vtg1 gene of Trust Sanger Institude zebra fish database prediction (ID: ENSDARG00000016825) designed the primer of sequence 2, sequence 3, and introduce HindIII and BamHI restriction enzyme site:

P1:5 '-GCTGCAAGCTTACTAATAGGAAGCCAACGAAGGACC-3 ' (sequence 2)

P2:5 '-CTCGGATCCGCTACAGTCAAGGCAAGCACAACAGC-3 ' (sequence 3)

Described primer is synthetic by match Parkson, Shanghai biotech company.

4. clone the zfvtg1-1.7 sequence

The clone of Zfvtg1-1.7 sequence carries out according to the method for " molecular cloning experiment guide ".

(1) amplification zfvtg1-1.7 sequence from genome

Get the zebra fish genomic dna of said extracted, use primer P1, P2 carries out pcr amplification, and reaction system is as follows:

Sterilization deionized water: 32 μ L

10 * PCR damping fluid: 5 μ L

dNTP(2.5mM each)： 8μL

P1： 1μL

P2： 1μL

Genomic dna: 2 μ L

Takara LA Taq archaeal dna polymerase (5U/ μ L) 1 μ L

Cumulative volume: 50 μ L

Reaction conditions:

94 ℃ 5 minutes, (94 ℃ 1 minute, 62 ℃ 1 minute, 72 ℃ 2 minutes) * 30 circulations, 72 ℃ 7 minutes.

Get the PCR product and carry out 1% agarose gel electrophoresis, the pulsating size of assay products.And with the above-mentioned PCR product of nucleic acid purification test kit purifying of U.S. promega company.

(2) make up the zfvtg1-1.7-EGFP recombinant plasmid

PCR product behind the purifying is with HindIII and BamHI double digestion, with same double digestion the pEGFP-1 carrier be connected, reaction system is as follows:

pEGFP-1： 1μL

PCR product: 4 μ L

SolI (containing ligase enzyme): 5 μ L

16 ℃ connect 3 hours, and transformed into escherichia coli competence DH5 α, extracting plasmid enzyme restriction identify, serve the order-checking of sea base health biotech company.This recombinant plasmid called after zfvtg1-1.7-EGFP.

Described pEGFP-1 carrier is available from Clontech company; Restriction enzyme is available from NEB company; Connect test kit fast available from TaKaRa company; DH5 α bacterial strain is preserved by this chamber.

The evaluation of embodiment 2 zfvtg1-1.7-EGFP promoter activities (spatial and temporal expression of EGFP and vtg1 is consistent)

1. make up the zfvtg1-1.7-EGFP transgenic zebrafish

2. the breed of zebra fish, transgenic technology and fluorescence observing method are with reference to " Zebrafish Book ".

(1) zebra fish is cultured

Wild-type zebra fish (Danio rerio) is available from international zebra fish resource center, and the zebra fish cultivating system is introduced from U.S. Aquatic Habitats company, and the nursing scheme is described according to " Zebrafish Book " and carried out.

(2) transgenosis

With restriction enzyme bgl II linearizing recombinant plasmid, be dissolved in behind the purifying and contain phenol red phosphate buffered saline buffer, concentration is 50ng/ μ L.When development of fertilized ova arrives one to two cell stage, under anatomical lens, carry out transgeneic procedure, it is 100pg (2nL) that every zygote changes the foreign gene amount over to.

(3) Fluirescence observation

Zygote behind the transgeneic procedure is divided three groups: blank group, group of solvents, EE2 treatment group (100ng/L), every group of 800 ovum.28 ℃ of constant temperature culture are regularly observed the luciferase expression situation under fluorescent microscope.

(4) RT-PCR and PCR identify the expression of EGFP and the integration in genome

Get the fish (5dpf, 15 every group) that observes the fish of fluorescence after the transgenosis and do not observe fluorescence respectively, by specification extracts total RNA with Trizol (Invitrogen) single stage method.Reverse transcription is a template with the total RNA of 1ug, the oligodT 1ul of 10mmol/L, 70 ℃, 5min; Add 10 * damping fluid 2ul on ice, the dNTP4ul of 25mmol/L, M-MLV (Invitrogen) 1ul (200U), 42 ℃, 60min, 95 ℃, 5min, reverse transcription are cDNA.Press document [the Aquat Toxicol.2003 Jan24 of Krovel AV and Islinger M etc.; 62 (2): 85-103; Biol Reprod.2002 Jun; 66 (6): 1881-92] design primer:

EGFP P1(5’-ACCACATGAAGCAGCACGACT-3’)，

EGFP P2(5’-CTTCTCGTTGGGGTCTTTGC-3’)；

vtg1 P1(5’-GCT GCT GCA TCTGTC AAT GT-3’)，

vtg1 P2(5’-CTG CTG CTG CTTTCA GAA GA-3’)。

With 1u reverse transcription product is template, and pcr amplification detects EGFP gene and vtg1 gene; Be that template is made positive control with the pEGFP-1 plasmid simultaneously.Reaction conditions is as follows: 94 ℃ of pre-sex change 5min, every circulation comprises 94 ℃ of sex change 40sec, 55 ℃ of renaturation 30sec, 72 ℃ are extended 40sec, totally 28 circulations, after last 1 loop ends again 72 ℃ extend 7min.Amplified fragments carries out the agarose gel electrophoresis analysis.

After the fish of waiting to observe fluorescence grows to maturation, extract genomic dna (promega DNA extraction test kit), PCR identifies the genome conformity of EGFP, primer is above-mentioned EGFP P1 and EGFP P2, simultaneously with plasmid pEGFP-1 be template as positive control, be that template is as negative control with wild-type fish genomic dna.Reaction conditions is: 94 ℃ of pre-sex change 5min, every circulation comprises 94 ℃ of sex change 40sec, 55 ℃ of renaturation 40sec, 72 ℃ are extended 1min, totally 28 circulations, after last 1 loop ends again 72 ℃ extend 7min.Amplified fragments carries out the agarose gel electrophoresis analysis.

2. in situ hybridization detects the expression of genetically engineered fish EGFP and vtg1

(1) preparation of recombinant plasmid pGEMTzfvtg1

Get the vtg1cDNA fragment (801bp) that above-mentioned experiment increases out, with the pGEM-T carrier reorganization of promega company, reaction system is as follows:

2 * ligase enzyme damping fluid: 5 μ L

pGEM-T(50ng)： 1μL

PCR product: 3 μ L

T4 dna ligase: 1 μ L

4 ℃ of connections are spent the night, transformed into escherichia coli competence DH5 α, the order-checking of extracting plasmid, this recombinant plasmid called after pGEMTzfvtg1.

(2) probe mark

Make the abundant linearizing of pGEMTzfvtg1 with restriction enzyme NcoI, reactions steps is as follows:

Reaction system:

pGEMTzfvtg1(1μg/μL)：30μL

10 * reaction buffer: 10 μ L

NcoI(10U/μL)： 8μL

Water: 52 μ L

Cumulative volume: 100 μ L

37 ℃ of reactions are spent the night.

Linearizing pGEMTzfvtg1 adopts the nucleic acid purification test kit purifying of promega company, and the concrete steps by specification carries out.

Adopting linearizing pGEMTzfvtg1 is template, carries out the rna probe mark with the NTP of digoxigenin labeled.Reaction system is as follows:

10 * SP6 RNA polymerase damping fluid: 2 μ L

DTT(50mM)： 2μL

Digoxigenin labeled NTP mixture (every kind of 2.5mM): 4 μ L

RNA enzyme inhibitors (40U/ μ L): 0.5 μ L

pGEMTzfvtg1(0.5μg/μL)： 1μL

SP6 RNA polymerase (50U/ μ L): 1 μ L

No RNA enzyme water: 9.5 μ L

37 ℃ were reacted 2 hours.

The DNA enzyme that adds 2 μ L (2U/ μ L) was hatched 30 minutes for 37 ℃.

Adding 2ul disodium ethylene diamine tetraacetate (pH8.0,0.2mol/L), 2.5ul lithium chloride (4mol/L), the 75ul dehydrated alcohol spends the night for subzero 20 degrees centigrade.4 ℃ 12000 rev/mins centrifugal 30 minutes, remove supernatant, with the water dissolution of the no RNA enzyme of 50ul.The sex change agarose gel electrophoresis is observed the quality of probe.Add the hybridization buffer of 450ul at last, standby.

(3) processing of juvenile fish and fixing

Juvenile fish is from fetal development first day beginning packet transaction: solvent (0.001% ethanol), 100ng/L EE2 changed liquid in per 24 hours.When growing into the 5th day, juvenile fish is collected in grouping.With 0.5 milliliter of PBST washed twice.0.5 milliliter of the Paraformaldehyde 96 of adding 4%, room temperature left standstill 5 minutes, removed Paraformaldehyde 96.Add 0.5 milliliter of 4% Paraformaldehyde 96 room temperature again and left standstill 5 minutes, place again 4 ℃ 12-15 hour.Remove Paraformaldehyde 96, once with 0.5 milliliter of washing of PBST.Use methanol wash again three times, it is frozen in subzero 20 degrees centigrade to add 0.5 ml methanol at last.

(4) hybridization

(1) with the solution shown in the table 1 and the time chien shih fixed embryo aquation again:

Table 1

The solution numbering	1	2	3	4
					PBST (milliliter) methyl alcohol (milliliter) washing time	1.5 4.5 5 minutes	3.0 3.0 5 minutes	4.5 1.5 5 minutes	6.0 05 minutes three times

(2) in PBST, remove chorion with the fine needle head;

(3) in room temperature with the protease K digesting embryo of 25 μ g/ml 30 minutes;

(4) room temperature was retightened the embryo 20 minutes with 4% Paraformaldehyde 96;

(5) PBST with 0.5ml washes 5 times, each 5 minutes;

(6) 65 ℃ of prehybridizations 2 hours in 300 μ L hybridization solutions, hybridization finish and placed 65 ℃ of preheatings 30 minutes with hybridization solution 1/20 dilution probe in preceding 30 minutes;

(7) remove prehybridization solution and change the hybridization solution that contains probe into, spend the night 65 ℃ of hybridization;

(8) remove hybridization solution, use table 2 solution 65 ℃ of washings.Solution will be 65 ℃ of preheatings 30 minutes, and every kind of solution washing time is 10 minutes.

Table 2

The solution numbering	1	2	3	4
					2 * SSC (milliliter) hybridization solution (milliliter) time	1.5 (25%) 4.5 (75%) 10 minute	3.0 (50%) 3.0 (50%) 10 minute	4.5 (75%) 1.5 (25%) 10 minute	6.0 (100%) 0 (0%) 10 minute

(9) wash twice with 0.5 milliliter of 0.2 * SSC at 65 ℃, each 30 minutes;

(10) with table 3 solution washing:

Table 3

The solution numbering	1	2	3	4
					PBST (milliliter) 0.2 * SSC (milliliter) time	1.5 (25%) 4.5 (75%) 5 minute	3.0 (50%) 3.0 (50%) 5 minute	4.5 (75%) 1.5 (25%) 5 minute	6.0 (100%) 0 (0%) 5 minute

(11) in PBST, add the BSA of 2mg/ml and 5% sheep serum as confining liquid, 4 ℃ of sealing embryos 1-3 hour;

(12) with the anti digoxin antibody of confining liquid 1/2000 dilution alkali phosphatase enzyme mark, make the embryo in this antibody-solutions 4 ℃ spend the night;

(13) wash each 15 minutes eight times with the PBST that contains 2mg/ml;

(14) with containing the sodium-chlor of 100mM Tris pH=9.5,100mM, magnesium chloride and the 0.1% soil temperature-20 solution equilibria embryo of 50mM;

(15) add alkaline phosphatase colour developing liquid chamber temperature colour developing 30 minutes to 1 hour, observations.

Hybridization related reagent of the present invention is available from Roche diagnostic reagent company.

Experimental result shows that in the system water of the female alcohol of 17 alpha-acetylenes that contains 100ng/L, tangible green fluorescence appears in the transgenic zebrafish liver that grows into the 5th day, and liver is expressed vtg1 simultaneously; And if do not contain the female alcohol of 17 alpha-acetylenes in the system water, fluorescence does not appear in the liver of transgenic zebrafish, does not have the expression of vtg1 yet.Therefore the EGFP expression of zfvtg1-1.7 startup is consistent with being expressed on the space-time of vtg1, and all is subjected to the regulation and control of oestrogenic hormon (the female alcohol of 17 alpha-acetylenes).

Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front should be understood that only to illustrate, but not limits the scope of the invention by any way.

From the above description, those skilled in the art can understand essential characteristic of the present invention at an easy rate, and under the situation that does not depart from its purport and scope, can carry out various changes and improvement to the present invention.

SEQUENCE LISTING

＜110〉Fudan University

＜120〉zebra fish vitellogenin-1 gene regulating sequence

<130>Fudan-20050619

<160>3

<170>PatentIn version 3.2

<210>1

<211>1720

<212>DNA

＜213〉zebra fish (Danio rerio)

<220>

<221>promoter

<222>(1)..(1720)

<400>1

actaatagga agccaacgaa ggactttaag caatgtgcaa ccaactggtt aaaagccttg 60

cagctgcatt cagcacaact tgcaatcaat ctacagatga ctatttagac aagtgtacag 120

aacattaaat agtccaaccg tgaagagata aaggaatgac aagcatctcc atcttttctt 180

atgaaaccat atttctagtt tttgcaatat tcctaagatg gaaaacactt gcaaacacag 240

attaaaacat agaactttat caaagatgac atccatattt ctgtttccac tgcaatttaa 300

gggtgacaga tacaaaaata ctcaatctct gggatgacat tataaaaatg caaaaatcta 360

catctcagtt ttgtcaggat taagttgaaa attgtcagcc atccatttgt taatggtgac 420

taaacaatcc tgcaagaact gtaagaaaag tacaatagca tatcatcagc atagcaatga 480

taagacacaa atttaaagct gctagtattg ttgcttagag aaagtgtata caaatgaaac 540

agaagagagc ctagcacaga aacttgaggc acgcacagga cagaggaaca gagtctgaca 600

tgtgagttcc tatggacaca gaaaaactcc tgtcaccaag ataggaggat aaccagagct 660

gtcccactat tcccaccaga gaatgcaatg tctttattaa agtgcagtga tcaacattgt 720

caaaatctac actaatatcc agcaaaacca gcacatacta attacctgca tgccattaaa 780

agatcatact taagacaact tggaacaaac aacaagggtc agtcacaaaa taccaggtct 840

accaaaccat ttacacccct tttttatttt ttccaaatac acaaacagtt gtcaaaatgt 900

acattatgtg gacaataaat acagttaaat atgacttggt gtaaataaaa gttaaaaagg 960

catgctgccc agctgtgttc tttcaccttt gcttataaca ggttattgta gntgtcttgt 1020

tgttacttat attaggccta ttttgtgtat atgccaaaaa ccttggccaa ttattaagta 1080

aaaaaaaata ttaggctaac attgacatca cttataaaaa ataatttctt aaaactcata 1140

catatttgcc ttagccccgg tagaaaaata attgcacagt gcctggttcc atacccatac 1200

catcaatggg atttatgcat tggttgctat gtaaatgaga tgttacatct acagtatgtt 1260

ctttaatttg tgttatcaga aaatcatgca ccaaaactta aaaacaaaaa atgcaccaaa 1320

aattatgtaa atctgcctgt ttagttaaat ctggccttaa atgtatacat aaaaataaat 1380

ttttaattaa ttacatctat ttataagtat ctatgtataa aacatctact gcatcagtat 1440

tattttatta ctgaaaaata ataacctttt tatatataaa agattggcga tcattttact 1500

gtgtacattg gcaaaaataa tgacatcatg acaggccagg ttaacctaac ctctggagat 1560

atttttgttc aaataaccaa gtcttaacct gtgtgcagtt ttttttttta cttgtttaaa 1620

gacttatgtt aataattaat ttaagaacta atacttggtc agcaagaaaa tcactataaa 1680

agacccgata ctctcaagag ttcttcacaa accaccagcc 1720

<210>2

<211>36

<212>DNA

＜213〉zebra fish (Danio rerio)

<400>2

gctgcaagct tactaatagg aagccaacga aggacc 36

<210>3

<211>35

<212>DNA

＜213〉zebra fish (Danio rerio)

<400>3

ctcggatccg ctacagtcaa ggcaagcaca acagc 35

Claims (5)

1, zebra fish vitellogenin-1 gene regulating sequence is characterized in that having the sequence of sequence 1, called after zfvtg1-1.7.

2, by the described zebra fish vitellogenin-1 of claim 1 gene regulating sequence, it is characterized in that described regulating and controlling sequence is by design primer amplification zebra fish genomic dna, obtain the dna sequence dna of 1720bp, described primer has the sequence of sequence 1 and sequence 2.

3, a kind of recombinant plasmid is characterized in that containing regulating and controlling sequence described in the claim 1 and enhanced green fluorescence protein gene, called after zfvtg1-1.7-EGFP.

4, claim 1 or 2 the zebra fish vitellogenin-1 gene regulating sequence purposes in preparation testing environment estrogen preparation.

5, the purposes of the recombinant plasmid of claim 3 in preparation testing environment estrogen preparation.