CN110592122B

CN110592122B - Zebra fish naalad2 gene promoter and application thereof

Info

Publication number: CN110592122B
Application number: CN201910758112.6A
Authority: CN
Inventors: 孙晨; 刘可春; 韩利文; 张磊; 何秋霞; 张云; 巴帅康; 高燕
Original assignee: Biology Institute of Shandong Academy of Sciences
Current assignee: Biology Institute of Shandong Academy of Sciences
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2021-04-13
Anticipated expiration: 2039-08-16
Also published as: CN110592122A

Abstract

The invention relates to a zebra fish naalad2 gene promoter and application thereof. The nucleotide sequence of the expression gene of the zebra fish naalad2 gene promoter is shown in SEQ ID No. 3. The invention discovers the zebra fish naalad2 gene promoter for the first time, and verifies the function and the starting efficiency of the promoter to discover that the regulation and control mode of the zebra fish naalad2 gene promoter is clearer and more controllable, and the establishment of the zebra fish naalad2 gene promoter has obvious advantages compared with promoters from other sources and has more application prospects compared with other related promoters.

Description

Zebra fish naalad2 gene promoter and application thereof

Technical Field

The invention relates to a zebra fish naalad2 gene promoter and application thereof, in particular to a zebra fish naalad2 gene promoter and a prostate disease research model constructed by using the promoter, belonging to the technical field of biotechnology and genetic engineering.

Background

Prostate cancer is one of the most common tumors that endanger male health. The incidence of the prostate cancer in China has been rising year by year in recent 20 years, the incidence of the prostate cancer in China in 2013 is 13.33/10 ten thousand, the annual average growth rate is as high as 9% -10%, and the incidence of the prostate cancer in men is 6 th. The onset age of the prostate cancer is lower before 55 years, the incidence rate gradually increases after 55 years, the incidence rate increases with the increase of the age, the peak age is 70-80 years, and the prostate cancer is a primary disease threatening the health of old men.

Until now, the research of the disease has been increasingly regarded by the nation and society. For the research of the prostate cancer disease, an animal model with operability, sensitivity, repeatability and stability is very important. Rodents such as mice or rats are common animal models for the study of prostate cancer diseases, but the animal models have high feeding cost and are inconvenient for genetic manipulation. In addition, when the prostate cancer disease is studied by using rodents, the organs of the rodents are generally dissected after the animals are sacrificed, and then the prostate tissues can be observed, so that the whole research period is long and the cost is high. The construction of a novel in vivo animal model applied to the research of the prostate cancer diseases is an urgent need in the field of the research of the prostate cancer diseases.

Zebrafish is a small tropical freshwater fish, can be used as a model organism for rapidly obtaining a large number of groups, and has the advantages of transparent embryos, in vitro development, easiness in genetic operation, mutant screening and the like. In 2005, whole genome sequencing of zebrafish was successful. On this basis, the reference genome of zebrafish was completed by the sanger institute in the united kingdom. Comparing the similarities and differences between the zebra fish and the human genome shows that the zebra fish and the human genome have 87% similarity, and about 70% of the human genes have at least one obvious zebra fish homologous gene. Zebrafish also exhibit high conservation in terms of signal pathway conduction, protein function and physiological structure, as compared to humans. Zebrafish have now been ranked by the national institutes of health as the third largest model organism, after humans and mice. Although fish do not have independent prostate tissue, studies have shown that prostate-like cells are present in fish gonads. Therefore, by combining with a transgenic technology, the construction of a novel zebra fish model is very suitable for the research of the prostatic cancer diseases.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a zebra fish naalad2 gene promoter and application thereof, and a prostate disease research model constructed by using a naalad2 gene promoter can solve the technical problems.

The technical scheme of the invention is as follows:

the nucleotide sequence of the expression gene of the zebra fish naalad2 gene promoter is shown in SEQ ID No. 3.

A recombinant plasmid constructed by inserting an expression gene of the zebra fish naalad2 gene promoter.

A recombinant cell inserted with the promoter expression gene of the zebra fish naalad2 gene or transformed with the recombinant plasmid.

The application of the expression gene of the zebra fish naalad2 gene promoter in constructing a prostate cancer animal model.

According to a preferred embodiment of the present invention, the steps of the application are as follows:

(1) taking a zebra fish genome as a template, and carrying out PCR amplification to prepare an expression gene of a zebra fish naalad2 gene promoter;

in the specific primer of the PCR amplification, the nucleotide sequence of an upstream primer is shown as SEQ ID NO.6, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 7;

(2) inserting the expression gene of the zebra fish naalad2 gene promoter prepared in the step (1) into pT2AL200R150G plasmid of a Tol2 transposon system to prepare recombinant plasmid;

(3) utilizing restriction endonuclease NotI to carry out enzyme digestion on transposon auxiliary plasmid pCS-TP in a Tol2 transposable system, and then synthesizing the mRNA of transposase by using the enzymatically digested transposon auxiliary plasmid pCS-TP as a template;

(4) introducing the recombinant plasmid prepared in the step (2) and the mRNA of the transposase prepared in the step (3) into AB-series zebra fish embryos together through microinjection, observing the zebra fish embryos subjected to microinjection by using an image acquisition tool, checking the expression condition of green fluorescent protein in the zebra fish embryos, and feeding the zebra fish embryos with fluorescence signals in vivo to sexual maturity to obtain F0-generation zebra fish;

(5) and (3) hybridizing the zebra fish of the F0 generation with the zebra fish of the AB line, detecting the fluorescent gene in the hybridized offspring by using an image acquisition tool, and screening to obtain the transgenic zebra fish with the prostate-like tissue specifically expressing fluorescence.

According to a further preferred embodiment of the present invention, in the step (1), the PCR amplification system is as follows:

according to a further preferred embodiment of the present invention, in the step (1), the PCR amplification procedure is as follows:

pre-denaturation at 94 ℃ for 15 s; denaturation at 98 ℃ for 10s, annealing at 57 ℃ for 10s, and extension at 72 ℃ for 30s for 30 cycles; final extension at 72 ℃ for 5min, then standing at 12 ℃ for 12 h.

According to a further preferred embodiment of the present invention, in the step (2), the specific steps of inserting the expression gene of the promoter of the naalad2 gene of zebrafish into the pT2AL200R150G plasmid of the Tol2 transposon system are as follows:

the expressed gene of the zebrafish naalad2 gene promoter was digested with restriction enzymes XhoI and HindIII, and ligated with pT2AL200R150G plasmid of the Tol2 transposon system, which was also digested with restriction enzymes XhoI and HindIII, by ligase.

More preferably, the restriction enzymes XhoI and HindIII are cut by the following system:

further preferably, in step (3), the restriction enzyme NotI is used to cleave the transposon helper plasmid pCS-TP in the Tol2 transposon system as follows:

according to a further preferred embodiment of the present invention, in the step (4), the image capturing means is a fluorescence microscope or a confocal laser microscope.

According to a further preferred embodiment of the present invention, in the step (4), the in vivo fluorescence signal is green spot-shaped and is specifically expressed in the gonad and intestinal tract tissues of the zebra fish.

According to a further preferred embodiment of the present invention, in the step (5), the screening criteria is to select zebrafish whose in vivo fluorescence signal is green dot-shaped and is specifically expressed in gonad and intestinal tract tissues.

The prostate cancer animal model constructed above is applied to screening of drugs for treating prostate cancer.

Advantageous effects

1. The invention discovers the zebra fish naalad2 gene promoter for the first time, and verifies the function and the starting efficiency of the promoter to discover that the regulation and control mode of the zebra fish naalad2 gene promoter is clearer and more controllable, and the establishment of the zebra fish naalad2 gene promoter has obvious advantages compared with promoters from other sources and has more application prospects compared with other related promoters;

2. the invention constructs a transgenic zebra fish model by utilizing the zebra fish naalad2 gene promoter for the first time, prostate-like tissues in gonads of the zebra fish model can be specifically marked by green fluorescence, and visualization of biological prostate-like tissues in a living body mode can be realized; in addition, the transgenic zebra fish model constructed by the invention provides a new experimental animal for the research of prostate related diseases and the research and development of medicaments thereof, and can improve the accuracy and reliability of related experiments.

Drawings

FIG. 1 is a photograph showing the homology alignment of five transcripts of the zebrafish naalad2 gene and the human PSMA gene at the protein level, wherein asterisks indicate that the amino acid residues at this position are completely conserved;

in the figure: a is the homology alignment between naalad2 and PSMA 201 transcript;

b is the homology alignment between naalad2 and PSMA 202 transcript;

c is the homology alignment between naalad2 and PSMA No. 203 transcript;

d is the homology alignment between naalad2 and PSMA 208 transcript;

e is the homology alignment between naalad2 and PSMA 212 transcript.

FIG. 2 shows the results of the expression of the male zebra fish naalad2 gene in five tissues, namely heart, liver, brain, gonad and intestinal tract:

in the figure: lane 1 is DNA Marker; lane 2 shows the expression of naalad2 in the gonads; lane 3 shows naalad2 expression in brain; lane 4 shows the expression of naalad2 in liver; lane 5 shows the expression of naalad2 in the gut; lane 6 is the expression of naalad2 in heart;

FIG. 3 is a picture of in vitro cloning of prostate specific promoter inserted into pMD-18T vector:

in the figure: a is a picture of a pMD-18T vector carrying prostate specific promoter in vitro cloning; wherein: lane 1 is DNA Marker;

lanes

2, 3, 4, 6 and 7 are pMD-18T vectors that do not carry prostate specific promoter clones; lane 5 is the pMD-18T vector carrying the prostate specific promoter clone in vitro;

b is a picture for verifying a pMD-18T vector carrying the prostate specific promoter in vitro clone by using two different restriction endonucleases; wherein: lane 1 is DNA Marker; lane 2 shows the restriction verification result of restriction enzyme DraI; lane 3 shows the restriction verification of the restriction enzyme NdeI.

FIG. 4 shows the results of the detection of prostate specific promoter activity; denotes embryo VS non-injected embryo after microinjection, p<0.01；^##Representing 48h embryo after microinjection 24h embryo after VS microinjection p<0.01。

FIG. 5 is a photograph of transgenic zebrafish of generation F0 based on prostate specific promoter constructed using the Tol2 transposon system; wherein the white arrow indicates the green fluorescence signal;

in the figure: a is a zebra fish picture 1 day after microinjection;

b is a zebra fish picture injected microscopically for 2 days;

c is a zebra fish picture injected microscopically for 6 days;

d is a picture of the wild AB line zebra fish fertilized for 1 day;

e is a picture of wild AB line zebra fish fertilized for 2 days;

f is a picture of wild AB line zebra fish fertilized for 6 days.

FIG. 6 is a photograph of transgenic zebrafish of generation F1; wherein the white arrows represent green fluorescent signals detected in the gonads; represents green fluorescent signal detected in brain;

in the figure: a is an F1 generation transgenic zebra fish picture fertilized for 1 day;

b is an F1 generation transgenic zebra fish picture fertilized for 2 days;

c is an F1 generation transgenic zebrafish picture fertilized for 5 days;

d is a picture of the wild AB line zebra fish fertilized for 1 day;

e is a picture of wild AB line zebra fish fertilized for 2 days;

f is a picture of wild AB line zebra fish fertilized for 5 days.

FIG. 7 is a photograph of gonad sites of prostate-like specific fluorescent transgenic zebrafish developing for 13 days;

in the figure: a is a picture of the gonad part of the transgenic zebra fish which is not treated by N-methyl-N-nitrosourea;

b is a picture of the gonad part of the transgenic zebra fish treated by the N-methyl-N-nitrosourea.

FIG. 8 is a histogram of fluorescence intensity statistics for transgenic zebrafish after N-methyl-N-nitrosourea treatment;

in the figure: denotes treatment VS control group, p < 0.01.

Detailed Description

The invention discloses a method for constructing a new model applied to the research of prostate diseases, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides a method for constructing a new model applied to the research of prostate diseases, wherein the used raw materials and reagents can be purchased from the market. The technical scheme of the invention is further illustrated by the following embodiments:

example 1: discovery of prostate-like specific promoter

1) The amino acid sequence of the PSMA gene was retrieved using the human genome database grch38.p12, then placed into the zebrafish reference genome database GRCz11, and after alignment it was initially judged that the homologous gene in zebrafish is an N-acetylated alpha-linked acidic dipeptidase 2 gene (N-acetylated alpha-linked acidic dipeptidase 2, nalad 2). In order to further confirm the homology between the zebrafish naalad2 gene and the human PSMA gene, the amino acid sequence of the zebrafish naalad2 gene and the amino acid sequence of the human PSMA gene were respectively aligned by using Clustal multiplex sequence alignment software, and the results are shown in FIG. 1. At the protein level, the naalad2 gene had 5 transcripts with similarity to the human PSMA gene of 67.53%, 67.12%, 66.81%, 67.19% and 66.38%, respectively.

2) RNA of five tissues such as liver, heart, brain, gonad and intestinal tract of the AB line male mature zebra fish of 4 months age are respectively extracted by using an RNA extraction kit of Novozan, and genome DNA is firstly removed by using a reverse transcription kit of the Novozan, the reaction temperature is 42 ℃, and the reaction time is 2 min. Which is subsequently reverse transcribed into cDNA. The five cDNAs are used as templates, and PCR technology is utilized to detect the expression condition of the zebra fish naalad2 gene in tissues. As shown in FIG. 2, naalad2 was expressed in higher amounts in brain, gonad, and intestinal tissues of zebrafish, but in lower amounts in liver and heart. The genome removal system is shown in table 1; the reverse transcription system is shown in Table 2; the reverse transcription reaction conditions are shown in Table 3; the PCR primer sequences are shown in Table 4; the PCR reaction system is shown in Table 5; the PCR reaction conditions are shown in Table 6.

TABLE 1 genome removal System

TABLE 2 reverse transcription System

TABLE 3 reverse transcription reaction conditions

TABLE 4 PCR primer sequences

Name (R)	Numbering	Sequence of
			Naalad2(L)	SEQ ID NO.4	agagaatattaaacatcatctcagg
Naalad2(R)	SEQ ID NO.5	gattagttttatttgggtatgaaag

TABLE 5 PCR reaction System

TABLE 6 PCR reaction conditions (30 cycles)

3) The Promoter Prediction software Promoter 2.0Prediction service is used for predicting that the Promoter of the naalad2 gene starts from the upstream-841 bp position of the 5' end of the first exon, and the nucleotide sequence is shown as SEQ ID NO. 1. The EPD Eukaryotic promoter database is used for carrying out secondary analysis on the positioning of the promoter, and the promoter is finally positioned at the position which is-462 bp upstream of the 5' end of the first exon, and the nucleotide sequence is shown as SEQ ID NO. 2.

4) The genome of a 4-month-old AB-line male sexual maturity zebra fish was extracted using an animal genome extraction kit from Tiangen corporation. And (3) amplifying the nucleotide sequence containing the predicted promoter in vitro by using the genome as a template and utilizing a PCR (polymerase chain reaction) technology. Subsequently, the in vitro amplification product was inserted into the pMD-18T vector using T4 ligase, and the above-mentioned modified pMD-18T vector was verified for enzyme digestion using two different restriction enzymes DraI and NdeI, respectively, as shown in FIG. 3. The PCR primer sequences are shown in Table 7; the PCR reaction system is shown in Table 8; the PCR reaction conditions are shown in Table 9.

TABLE 7 PCR primer sequences

Name (R)	Numbering	Sequence of
			Promoter(L)	SEQ ID NO.6	ACGGACGGCTAAAACAGAGT
Promoter(R)	SEQ ID NO.7	ACGAATACAGGTGCGGAGA

TABLE 8 PCR reaction System

TABLE 9 PCR reaction conditions (30 cycles)

Example 2: activity detection of prostate-like specific promoters

The promoter sequence was excised from the engineered pMD-18T vector using restriction enzymes SacI and HindIII and inserted into the pGL3-Basic vector using T4 ligase. pGL3-Basic vector linked with promoter was introduced into AB line zebra fish embryos by microinjection technique. After microinjection is carried out for 24 hours and 48 hours, the content of the firefly enzyme in the zebra fish body is detected by using firefly enzyme reporter gene detection kits, and the result is shown in figure 4. After 24h or 48h of injection, positive signals of firefly enzyme were detected in both promoter-injected groups compared to the non-injected group embryos, and the firefly enzyme activities were statistically different compared to the non-injected control group. In addition, after the activity of the firefly enzyme in the zebra fish 24h after injection and the activity of the firefly enzyme in the zebra fish 48h after injection are compared, the activity of the firefly enzyme in the zebra fish 48h after injection is obviously higher than that in the zebra fish 24h after injection. The above results indicate that the prostate-like specific promoter has an activity of promoting gene expression in zebrafish, and that the promoter activity increases as the development time of zebrafish increases.

The detection proves that the expression characteristics of the zebra fish naalad2 gene and the human PSMA gene are remarkably different, the zebra fish naalad2 gene has higher expression level in brain, gonad and intestinal tissues of zebra fish, but has lower expression level in liver and heart, the human PSMA gene is mainly expressed in organs such as adult prostate, gonad and liver, and the PSMA gene is not expressed in fetal period of human, so that the regulation and control mode of the naalad2 gene promoter is more clear and controllable, and the application prospect of the promoter is better than that of other related promoters when the promoter is used as a related model.

Since the promoter of the present application is derived from zebrafish, the promoter can be applied to the construction of zebrafish models. Correspondingly, other prostate-specific promoters known at present are derived from human or rodent species, such as mouse and rat. Therefore, they can only be applied to the construction of models such as cells, mice or rats, and cannot be applied to zebra fish. Zebrafish are a novel vertebrate model organism between cells and rodents, which combine the advantages of both cells and rodents. In the field of biological medicine research, the zebra fish model has the advantages of rapidness, simplicity, convenience, high throughput and the like of a cell model, and also has the advantages of reliability, comprehensiveness, high connotation and the like of rodents. Therefore, in the field of animal model construction, the promoter disclosed in the application has greater practical value compared with other prostate-specific promoters. In addition, in the field of biomedical research, the promoter has a greater application prospect.

Example 3: application of prostate-like specific promoter

The promoter sequence was excised from the engineered pMD-18T vector using restriction enzymes XhoI and HindIII and inserted into the pT2AL200R150G plasmid of the Tol2 transposon system using T4 ligase. Transposase mRNA was co-introduced into AB-line zebrafish embryos with pT2AL200R150G plasmid inserted into the promoter using the Tol2 transposon system in conjunction with microinjection. After microinjection for 1, 2 and 6 days, the expression of green fluorescent protein in zebrafish embryos was examined under a fluorescence microscope, respectively, and the results are shown in fig. 5. Fluorescent signals can be detected in the bodies of the zebra fish after microinjection for 1, 2 and 6 days.

Zebrafish embryos with fluorescent signals in vivo were fed to sexual maturity and crossed with AB line zebrafish. When the progeny developed to

days

1, 2 and 5, their in vivo fluorescence expression was examined under a fluorescence microscope, respectively, and the results are shown in FIG. 6. When the progeny of the cross developed to 1, 2 and 5 days, a green fluorescent signal was detectable in vivo.

Example 4: application of prostate-like specific fluorescent transgenic zebra fish

1. Acquisition of transgenic Zebra Fish

Healthy sexually mature prostate-like specific fluorescent transgenic zebra fish are cultured according to the weight ratio of male and female 1: 2, placing the mixture into a blending tank, placing a partition plate in the middle, placing the mixture in a dark environment, taking out the partition plate before lighting the next day, and lighting to stimulate the mixture to ovulate. Collecting fertilized eggs, washing the fertilized eggs with new zebra fish embryo culture water for 3 times, and disinfecting and cleaning the fertilized eggs; then, the fertilized eggs are transferred into clean water for culturing zebra fish, 0.2ppm of methylene blue is added into the culture water, the culture is carried out under the condition of light control at 28 ℃ for 14h of light/10 h of dark cycle, 1/3 of water is changed every 24h in the middle, and dead embryos are sucked out in time.

2. Treatment of compounds

Healthy fluorescent transgenic zebra fish which developed to day 13 were selected and placed in 6-well plates with 10 fish per well. 1 blank control group, 1N-methyl-N-nitrosourea treatment group (6mL of culture water to which a final concentration of 10. mu.M/mL of N-methyl-N-nitrosourea solution was added) was set. And (3) putting the two groups of zebra fishes into a constant-temperature incubator at 28 ℃ respectively, and continuously culturing for 2 hours.

3. Experimental results collection and data analysis

After the compound treatment is finished, the two groups of zebra fish are anesthetized by using a 0.02 mass percent tricaine solution. The green fluorescence photograph of the gonad part of the zebra fish was taken under a fluorescence microscope, and the result is shown in fig. 7. According to the collected pictures, the fluorescence intensity of the transgenic zebra fish is calculated by using Image-Pro Plus software, and the fluorescence intensities of the two groups of zebra fish are counted by using GraphPad prism5.0 software, and the result is shown in FIG. 8.

N-methyl-N-nitrosourea is a common chemical mutagen for prostate cancer and can be used for constructing an animal model of human prostate cancer. When 10 mu M/mL of N-methyl-N-nitrosourea is used for continuously treating the prostate-like specific fluorescent transgenic zebra fish for 2 hours, the fluorescence intensity of the zebra fish is obviously enhanced. The results indicate that the promoter of the zebra fish naalad2 gene has functional similarity with the promoter of the human PSMA gene, and the promoters have high expression in prostatic cancer tissues and can be used as potential diagnosis indexes of prostatic cancer. The fluorescent transgenic zebra fish constructed based on the promoter of the naalad2 gene can be applied to the construction of prostate cancer animal models and the research of prostate cancer diseases.

Sequence listing

<110> institute of biological research of academy of sciences of Shandong province

<120> zebra fish naalad2 gene promoter and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 961

<212> DNA

<213> Zebra fish (Barchydanio rerio var)

<400> 1

gaccagcctg gttttagagg ttttttggcc acttccaggc tggtttccag tcatttccag 60

cctggtctta gctggtttgg ctggaaagtg acctgctaaa accagcttga ccagcctggt 120

ttaagctgga catagctggt tttggctggg gtcccagcct ggctaggttg gtcaagctgg 180

ttttagctgg tcatttctca gcttgtacag ctaaaaccag gctggaaatg gctggaaacc 240

agcctggaag tggccaaaac ccctctaaaa ccagcctggt caaccagcta aaaccagcca 300

accatcctag gctggtttaa gctggatttt tcagcaggaa atcttcctgt tttaaagaga 360

tggcagggaa aaatggaatt taatgcagtg cttcttgtct gagactcact ttatattaaa 420

tatctattag gcattgaaga tcacttattt ttaaaataaa cagatcttaa acgtggcaat 480

tttggaaaga cagttcacag gaagccgttg tgctggctga aaattaaaag tctgtttgca 540

tataccccat tagtaagttc ccatcctcaa aggataggtc aggttttttt tactcattag 600

aaaaacaaat catcattatg ttcttcttct gttaatttaa aaggtcattt tcagcagcaa 660

ccaacagcca aatatttatc tcacttcata aacctgtgaa atttctgcac ggcgacatct 720

atgaacgaac ccttttacgc tcgctaaatc aagtgaattg gtgaatcatg ttcactcaaa 780

cgaaccgatt cgctgaaatg aactaaccca aaccaaaaca cgcctacatc actctccccc 840

cacagacagt attagctctc caactttcaa cgagttatga gcggcgttta gtcaaatggg 900

ttctgaggat aaaataatcc gctggatctg ccggctaaca gccattacag ctttcttctt 960

g 961

<210> 2

<211> 582

<212> DNA

<213> Zebra fish (Barchydanio rerio var)

<400> 2

ttaatgcagt gcttcttgtc tgagactcac tttatattaa atatctatta ggcattgaag 60

atcacttatt tttaaaataa acagatctta aacgtggcaa ttttggaaag acagttcaca 120

ggaagccgtt gtgctggctg aaaattaaaa gtctgtttgc atatacccca ttagtaagtt 180

cccatcctca aaggataggt caggtttttt ttactcatta gaaaaacaaa tcatcattat 240

gttcttcttc tgttaattta aaaggtcatt ttcagcagca accaacagcc aaatatttat 300

ctcacttcat aaacctgtga aatttctgca cggcgacatc tatgaacgaa cccttttacg 360

ctcgctaaat caagtgaatt ggtgaatcat gttcactcaa acgaaccgat tcgctgaaat 420

gaactaaccc aaaccaaaac acgcctacat cactctcccc ccacagacag tattagctct 480

ccaactttca acgagttatg agcggcgttt agtcaaatgg gttctgagga taaaataatc 540

cgctggatct gccggctaac agccattaca gctttcttct tg 582

<210> 3

<211> 2471

<212> DNA

<213> Zebra fish (Barchydanio rerio var)

<400> 3

acggacggct aaaacagagt cggtttgcac acaattacaa cattagttta atataacatt 60

attagtgatt ttatttcacc caaaatgttc atttgaatta atacttcatt aaaaatcaag 120

ccgcgtttat ccgtataaat tacgagcgtt caaacactga actattgact cctattcgcc 180

aaagactcgt tcatctgaat catttaatgt tgacgattca attgaaccaa aacacggcgt 240

ttttggacat ttaaaagaac aaccgtgttc atatggaata atatagctta attaataaag 300

cctagaacaa tgtaatctaa gaagagagag aaatattcaa aataaaactt tgttttgaga 360

taatatgtgt ttgtatttta tatatatgta tatgttatag gctatacaca tagacacaca 420

cacaaaactc cttcatttat tcattttcct tcagtttagt ccctttattc atcaggggtt 480

gccacggcag aatgaaccgc cgacttatcc aacatatgtt tcatgcagcg gatgccattc 540

cagcagcaac ccaacactgg gaaacaccca tacacactca ttcacactca taaactacgg 600

acaatttaga ttattcaatt tacctatagc gcatgtgttt ggactgtggg ggaaaccgga 660

gtcacccgga ggaaactcca gctaccttct tgcagcaacc atcttgctgt gctacccact 720

gcgccaccgt gatgccaata aatgtacaca cacacacaca cgcacacaca cacacacaca 780

cgcacacaca cacacacacg cacacacaca cacacacaca cacacacaca cacacacaca 840

cacatatata tataaataaa taatttttgg cagcactaaa ttaaacttca ctcaaaaata 900

attttaaaaa ggtgtcatta ttaactctcc ctttaactct gtttcaaacc tgtatgagtt 960

tcattctatt aaaaacaaaa taagctattt tgactaaatt aattgttaca agtttccaac 1020

atttttcaaa atatcttctt ttgtgttcaa cagaaaataa ggaaattgtg acttggggtg 1080

agtaaatatg aagtattgtt tttatttatt atttattgtt tgttttattt ggggtgaact 1140

atccctttta gagttaacat atctactaaa aatacctgct gtgtttctca gtaaatggta 1200

aacttactgt gaaagatttc tgtgactatt taaagtttta taaatttaat tttacagcat 1260

cgacatcata atgtaattaa aatacaacca gttaaataaa cttaagcatt catttagttg 1320

ttcaagcgta aaacgacatg atagactgtg taaccgctag cgccattcgg tagcaaggta 1380

gcccagaaac tccaatactg aggtaaaata accctgctga aaaatccagc ttaaaccagc 1440

cttagatggt tggctggttt tagctggtcg accagcctgg ttttagaggt tttttggcca 1500

cttccaggct ggtttccagt catttccagc ctggtcttag ctggtttggc tggaaagtga 1560

cctgctaaaa ccagcttgac cagcctggtt taagctggac atagctggtt ttggctgggg 1620

tcccagcctg gctaggttgg tcaagctggt tttagctggt catttctcag cttgtacagc 1680

taaaaccagg ctggaaatgg ctggaaacca gcctggaagt ggccaaaacc cctctaaaac 1740

cagcctggtc aaccagctaa aaccagccaa ccatcctagg ctggtttaag ctggattttt 1800

cagcaggaaa tcttcctgtt ttaaagagat ggcagggaaa aatggaattt aatgcagtgc 1860

ttcttgtctg agactcactt tatattaaat atctattagg cattgaagat cacttatttt 1920

taaaataaac agatcttaaa cgtggcaatt ttggaaagac agttcacagg aagccgttgt 1980

gctggctgaa aattaaaagt ctgtttgcat ataccccatt agtaagttcc catcctcaaa 2040

ggataggtca ggtttttttt actcattaga aaaacaaatc atcattatgt tcttcttctg 2100

ttaatttaaa aggtcatttt cagcagcaac caacagccaa atatttatct cacttcataa 2160

acctgtgaaa tttctgcacg gcgacatcta tgaacgaacc cttttacgct cgctaaatca 2220

agtgaattgg tgaatcatgt tcactcaaac gaaccgattc gctgaaatga actaacccaa 2280

accaaaacac gcctacatca ctctcccccc acagacagta ttaagctctc caactttcaa 2340

cgagttatga gcggcgttta gtcaaatggg ttctgaggat aaaataatcc gctggatctg 2400

ccggctaaca gccattacag ctttcttctt gatcggcttt attattggtg agtctccgca 2460

cctgtattcg t 2471

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

agagaatatt aaacatcatc tcagg 25

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gattagtttt atttgggtat gaaag 25

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

acggacggct aaaacagagt 20

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

acgaatacag gtgcggaga 19

Claims

1. The nucleotide sequence of the expression gene of the zebra fish naalad2 gene promoter is shown in SEQ ID No. 3.

2. A recombinant plasmid constructed by inserting an expression gene of the promoter of the naalad2 gene of zebrafish of claim 1.

3. A recombinant cell into which a promoter-expressing gene of the naalad2 gene of zebrafish of claim 1 is inserted or which is transformed with the recombinant plasmid of claim 2.

4. Use of the expression gene of the zebra fish naalad2 gene promoter of claim 1 in constructing an animal model of prostate cancer.

5. Use according to claim 4, characterized in that the steps are as follows:

6. The use of claim 5, wherein in step (1), the PCR amplification system is as follows:

7. the use of claim 5, wherein in step (1), the PCR amplification procedure is as follows:

8. The use as claimed in claim 5, wherein in step (2), the insertion of the expressed gene of the promoter of the naalad2 gene of zebrafish into the pT2AL200R150G plasmid of the Tol2 transposon system is carried out by the following steps:

9. The use of claim 8, wherein in step (2), the restriction enzymes XhoI and HindIII are cut as follows:

10. the use as claimed in claim 5, wherein in step (3), the restriction enzyme NotI cleaves the transposon-helper plasmid pCS-TP in the Tol2 transposon system as follows:

11. the use of claim 5, wherein in step (4), the image acquisition means is a fluorescence microscope or a confocal laser microscope.

12. The use of claim 5, wherein in step (4), the in vivo fluorescence signal is green spot-shaped and is specifically expressed in gonads and intestinal tissues of zebrafish.

13. The use of claim 5, wherein in step (5), the screening criteria is to select zebrafish whose in vivo fluorescence signal is green punctate and specifically expressed in gonadal and intestinal tissues.

14. Use of the prostate cancer animal model constructed according to claim 4 for screening a medicament for treating prostate cancer.