CN114591962A

CN114591962A - Promoter of lgals3bpb gene and application of promoter in construction of zebra fish model

Info

Publication number: CN114591962A
Application number: CN202210457052.6A
Authority: CN
Inventors: 李礼; 贺蒋勇; 赵方莹; 陈冰玥; 崔念飞; 李智繁
Original assignee: Chongqing Institute of Green and Intelligent Technology of CAS
Current assignee: Chongqing Institute of Green and Intelligent Technology of CAS
Priority date: 2022-04-27
Filing date: 2022-04-27
Publication date: 2022-06-07

Abstract

The invention relates to the technical field of a nerve cell marking method and related animal models, in particular to a promoter of an lgals3bpb gene and application thereof in construction of a zebra fish model. The application of the promoter of the lgals3bpb gene in constructing the zebra fish model comprises the following steps of: obtaining an lgals3bpb gene promoter through PCR reaction; constructing an expression vector regulated by an lgals3bpb gene promoter; and transferring the expression vector into fertilized eggs of the zebra fish, and performing culture, screening and multi-generation hybridization to obtain the stably inherited transgenic zebra fish strain. The technical scheme can solve the technical problem that a zebra fish animal model capable of positioning and operating microglia is absent in the prior art, creates conditions for researching the microglia, researching disease action mechanism, screening drugs and testing drug effect, and has good application prospect.

Description

Promoter of lgals3bpb gene and application of promoter in construction of zebra fish model

Technical Field

The invention relates to the technical field of a nerve cell marking method and related animal models, in particular to a promoter of an lgals3bpb gene and application thereof in construction of a zebra fish model.

Background

Zebrafish (Danio rerio) is a common tropical fish, and a zebrafish cell marker technology, a tissue transplantation technology, a mutation technology, a haploid breeding technology, a transgenic technology, a gene activity inhibition technology and the like are mature, and thousands of zebrafish embryonic mutants are good resources for researching embryonic development molecular mechanisms, and some zebrafish embryonic mutants can also be used as human disease models. Because the similarity of the zebra fish gene and the human gene reaches 87 percent, the result obtained by performing a drug experiment on the zebra fish gene is also suitable for the human body in most cases; the zebra fish embryo is transparent, so biologists can easily observe the influence of the medicine on the internal organs of the zebra fish; female zebra fish can lay eggs for 200, and embryos can grow and form within 24 hours, so that biologists can perform different experiments on the same generation of fish, further study the pathological evolution process and find out the causes of diseases.

Microglia (microglia) are tissue-resident macrophages of the central nervous system whose roles include clearing cellular debris, signaling neurons and astrocytes to mediate synaptic connections, and maintaining homeostasis of the overall nervous system. It is necessary to fully research microglia, understand the mechanism of occurrence and development, and have important significance for the research of related diseases. Due to the above-mentioned advantages of the zebrafish model, more and more researchers are using the zebrafish model to study microglia. The zebra fish is used as an animal model to carry out experimental research on microglia, and the requirement that the microglia in the living body of the zebra fish can be specifically marked and operated is firstly needed, so that higher requirements are provided for research means and methods. Although, the present studies have revealed some genes specifically expressed in microglia, theoretically, the promoters of these genes can be used to control some effector genes to produce specific effects in microglia.

At present, only two genes, namely apoeb and ccl34b.1, are used for marking microglia, transgenic strains based on the two genes are obtained by a BAC (Bacterial artificial chromosome) mediated transgenic technology, but a reliable promoter sequence for marking the microglia is not determined yet, and the reliable promoter sequence cannot be randomly operated according to the research purpose, so that the research work is greatly limited. The length and distribution of different gene promoters are different, and promoter sequences with ideal performance are difficult to effectively capture in specific expression genes of a large number of microglia. And in some cases, even after capturing the promoter, it is difficult for the promoter to efficiently drive the expression of the effector gene in the zebrafish animal model.

In conclusion, it is highly desirable to construct a zebrafish animal model capable of locating and manipulating microglia to meet the requirements of disease action mechanism research, drug screening and drug efficacy test.

Disclosure of Invention

The invention aims to provide application of a promoter of an lgals3bpb gene in constructing a zebra fish model, so as to solve the technical problem that the zebra fish model capable of positioning and operating microglia is lacked in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

an application of a promoter of an lgals3bpb gene in constructing a zebra fish model comprises the following steps of:

s1: obtaining an lgals3bpb gene promoter through PCR reaction; the nucleotide sequence of the promoter of the lgals3bpb gene is shown in SEQ ID NO. 1;

s2: integrating the promoter of the lgals3bpb gene on a vector to obtain an expression vector; the lgals3bpb gene promoter is used for controlling the expression of a fluorescent protein gene;

s3: transferring the expression vector into fertilized eggs of the zebra fish, and culturing and screening to obtain F0-generation zebra fish; and hybridizing the F0-generation zebra fish and the wild zebra fish for multiple generations to obtain the stably inherited transgenic zebra fish strain.

The scheme also provides a promoter of the lgals3bpb gene, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

The principle and the advantages of the scheme are as follows:

in the technical scheme, a promoter fragment (4141bp) of the lgals3bpb gene is obtained by a DNA polymerase chain reaction; and then constructing an expression vector, transgenosis to zebra fish fertilized eggs, and finally hybridizing for multiple generations to obtain a stable transgenic fish line.

In microglia, there are some genes that are expressed in an enriched manner, such as apoeb gene (apolipoprotein E) and ccl34b.1 gene (chemokine (C-C motif) ligand 34b, duplicate 1). The two genes are specifically expressed in microglia, and are considered to be microglia marker genes by scientific researchers. The inventor finds that various genes such as lgals3bpb gene and the like are enriched and expressed in microglia by sequencing genes in the microglia and researching expression mode. Further studies found that the expression pattern of the lgals3bpb gene coincided with that of the two marker genes of known microglia. The inventors have utilized the above findings to further study the expression regulation and control mode of genes that are enriched and expressed in microglia, and have cloned and analyzed the promoter sequences of these genes. It was found that although there are a large number of candidate genes, the promoter sequences of these candidate genes related to the development process of microglia are very difficult to clone. Although some high expression genes can be obtained by cloning through technical means, when the partial sequences are connected with fluorescent protein genes, the expression of the fluorescent protein cannot be started in zebra fish bodies. The inventor obtains a promoter of the lgals3bpb gene through a large amount of screening, constructs an expression vector by using the promoter and then constructs a transgenic zebra fish model. The promoter of the lgals3bpb gene can promote the specific expression of the fluorescent protein gene in the microglia of the zebra fish model, thereby realizing the labeling of the microglia and the observation of the microglia. If the fluorescent protein is replaced by other effector genes, the function of the effector gene can be realized. In the prior art, the research of marking microglia by using the lgals3bpb gene or a promoter thereof is not reported, and a promoter sequence which is used for specifically marking the microglia of the zebra fish and can be operated is not reported. The carrier containing the lgals3bpb gene and the fluorescent protein gene is transferred into the zebra fish body, and the zebra fish transgenic line constructed by using the lgals3bpb gene promoter is a very good zebra fish animal model for researching microglia, can be used for mechanism research of the action of the microglia in neurodegenerative related diseases, and can also be used for drug screening and drug effect test.

Further, in S1, the primers for the PCR reaction include:

F1-lgals3bpb：5’-tcgaattcctgcagcccgggTGTTGCTCTGAAGTCAGAGG-3’；

R1-lgals3bpb：5’-GCTGTTGTACGTCCAAGTACCATTCATACTATTCACATTT-3’；

F2-lgals3bpb：5’-GTACTTGGACGTACAACAGC-3’；

R2-lgals3bpb：5’-tcctcgcccttgctcaccatGATGTCAGAAACAGCAACGA-3’。

the primers can be used for realizing the effective amplification of target genes.

Further, in S3, the way to obtain zebrafish F0 generation is: simultaneously microinjecting the expression vector and the transposase into fertilized eggs of the zebra fish; selecting fertilized eggs expressing the fluorescent protein 3 days after fertilization; and culturing until sexual maturity to obtain F0 generation zebra fish. Integrating a target gene into a zebra fish genome in a transgenic mode, and screening to obtain the transgenic zebra fish with the target gene transferred.

Further, in S3, the DNA template of the transposase has the sequence shown in SEQ ID NO. 9. The transposon is a sequence capable of changing its position and comprises an intermediate sequence and a terminal directional repeat sequence, wherein the intermediate sequence can express transposase. The transposase in the scheme is a tool for improving the plasmid insertion efficiency, and if the transposase is not used, the insertion of the foreign gene on the zebra fish genome can be carried out, but the efficiency is reduced.

Further, in S3, the culture temperature was 28.5 ℃. The culture temperature is the optimum temperature for culturing zebra fish.

Further, in S3, crossing the F0 generation zebra fish with the wild zebra fish, screening the offspring expressing the fluorescent protein, and obtaining the F1 generation zebra fish; and mating the F1-generation zebra fish with wild zebra fish to obtain the stably inherited transgenic zebra fish strain.

Further, in S1, the PCR reaction system includes: 18. mu.L of water, 25. mu.L of buffer, 0.5. mu.L of DNA polymerase, 1.5. mu.L of template, 2.5. mu.L of forward primer and 2.5. mu.L of reverse primer. The reaction system can be used for effectively amplifying the target DNA segment.

Further, in S2, the sequence of the fluorescent protein gene is shown in SEQ ID NO. 6. The green fluorescent protein is a marker protein commonly used in the field, is regulated and controlled by an lgals3bpb gene promoter, can enable microglia to generate a fluorescent signal so as to be distinguished from other types of cells, and is convenient for observing the microglia.

Furthermore, the promoter of the lgals3bpb gene is used for controlling the expression of the fluorescent protein in microglia of the zebra fish.

The scheme provides an operable promoter sequence for marking the microglia, and the microglia can be operated through the promoter specificity. For example, the promoter carries a nitroreductase gene which can kill microglia particularly but not damage other macrophages; or a certain gene is supplemented or knocked out in microglia cells or Langerhans cells by utilizing the specificity of the promoter; fluorescent proteins can also be incorporated behind the promoter for localizing microglia.

In conclusion, the inventor discovers that immune cells in the brain of zebra fish can be divided into a plurality of groups through single cell sequencing analysis, screens a plurality of genes with similar expression patterns to apoeb genes and ccl34b.1 genes (microglia marker genes acknowledged in the prior art), and uses the genes as candidate genes of cloned promoters. And apoeb gene and ccl34b.1 gene, although expressed in large amounts in microglia, their expression was not concentrated in immune cells of brain. In the case of apoeb gene, it is also expressed in a certain amount in neuronal cells. Therefore, it is necessary to select a gene capable of being specifically expressed in microglia.

Due to the complexity of the core promoter sequence with specificity for cellular expression, the inventors have obtained only a portion of the sequence upstream of the start codon of the candidate gene by extensive attempts. The sequences upstream of these obtained initiation codons were integrated into expression plasmids and transgenic into zebrafish, and not all of the cloned sequences upstream of the initiation codons were found to have activity to initiate effector gene expression. Finally, only the sequence upstream of the initiation codon of the cloned apoc1 gene and the sequence upstream of the initiation codon of the lgals3bpb gene were found to initiate the expression of the effector gene, i.e., the core promoter sequence was obtained. In further experiments, the promoter of the apoc1 gene and the promoter of the lgals3bpb gene can promote the specific expression of effector genes in microglia, and the effector genes can not be expressed in neurons and other immune cells except the microglia. This effect makes it possible to observe and manipulate microglia specifically. The expression patterns of the apoc1 gene and the lgals3bpb gene, and the discovery of the promoter of the apoc1 gene and the promoter of the lgals3bpb gene obtain unexpected technical effects, and overcome the technical problem of solving the problem of lacking an animal model capable of positioning and operating microglia in the prior art.

In addition, the promoter-tagged transgenic line of the lgals3bpb gene was more fluorescent than the promoter-tagged transgenic line of the apoc1 gene. Furthermore, the promoter marker of the apoc1 gene had a larger range than that of the lgals3bpb gene, and the promoter of the lgals3bpb gene had a relatively high specificity.

Drawings

FIG. 1 is an electrophoretogram of a promoter fragment clone of the lgals3bpb gene of example 1.

FIG. 2 is a schematic diagram showing the construction process of pT2AL-cryaa-lgals3bpb-eGFP plasmid in example 1.

FIG. 3 is a schematic diagram of pT2AL-cryaa-lgals3bpb-eGFP plasmid of example 1.

FIG. 4 is a PCR electrophoretogram of the colony of example 1.

FIG. 5 is a schematic diagram of the construction and screening of transgenic zebrafish of example 1.

FIG. 6 is a crya-cerulean marker observation of 3dpf for example 1.

FIG. 7 is fluorescence microscopy images of zebrafish Tg (lgals 3bpb: eGFP) whole body eGFP positive signal detection 3 days after fertilization in example 1.

FIG. 8 is the results of co-localization of transgenic zebrafish eGFP and mesencephalic pan microglia 3 days after fertilization in example 1.

FIG. 9 shows the co-localization of eGFP and the mesencephalon pan-microglia in the transgenic lines of example 1 at 12 months of development.

FIG. 10 shows the results of the study of the expression pattern of the lgals3bpb gene of example 2 and the marker gene of known microglia.

FIG. 11 shows the expression of green fluorescent protein regulated by the Apoeb gene promoter in the prior art study of example 2.

FIG. 12 is a fluorescence microscopy image of Zebra fish Tg (coro1a: DsRed; vmp1-eGFP) line of example 2.

FIG. 13 is a fluorescence micrograph of a Zebra fish Tg (coro1a: DsRed; havcr1-eGFP) line of example 2.

FIG. 14 is a fluorescence microscopy image of full-body eGFP positive signal detection of zebrafish Tg (apoc 1: eGFP) 3 days after fertilization in example 3.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used therein are commercially available.

Example 1: construction of zebra fish animal model

S1: cloning of the promoter of the lgals3bpb Gene

The promoter of the lgals3bpb gene (SEQ ID NO.1, 4141bp) was PCR amplified using the following primers:

F1-lgals3bpb：5’-tcgaattcctgcagcccgggTGTTGCTCTGAAGTCAGAGG-3’，SEQ ID NO.2；

R1-lgals3bpb：5’-GCTGTTGTACGTCCAAGTACCATTCATACTATTCACATTT-3’，SEQ ID NO.3；

F2-lgals3bpb：5’-GTACTTGGACGTACAACAGC-3’，SEQ ID NO.4；

R2-lgals3bpb：5’-tcctcgcccttgctcaccatGATGTCAGAAACAGCAACGA-3’，SEQ ID NO.5。

the PCR system (using the PCR kit of Hippocampus Biotech (Shanghai) Co., Ltd.) was:

water: 18 mu L of the solution; 2 × heiff buffer: 25 mu L of the solution; heiff enzyme: 0.5 mu L; genomic template (zebrafish genomic DNA): 1.5 mu L; f-terminal primer (10. mu.M): 2.5 mu L; r-terminal primer (10. mu.M): 2.5. mu.L.

The PCR procedure was:

98℃，3min，×1；

(98℃、10s)→(60℃、20s)→(72℃、2min)，×40；

72℃，5min，×1。

the promoter amplification product of the purified and enriched lgals3bpb gene is obtained by the conventional agarose electrophoresis and gel cutting recovery means in the prior art, and is sent to a third party company for sequencing to confirm that the recovered product is a target sequence. The electrophoretogram of the two DNA fragments is shown in FIG. 1.

The following primers were used for PCR amplification of eGFP fluorescent protein (SEQ ID NO. 6):

F1-eGFP：5’-atggtgagcaagggcgagga-3’，SEQ ID NO.7；

R1-eGFP：5’-tggatcatcatcgataggcctttacttgtacagctcgtcca-3’，SEQ ID NO.8。

water: 18 mu L of the solution; 2 × heiff buffer: 25 mu L of the solution; heiff enzyme: 0.5 mu L; plasmid template: 1.5 mu L; f-end primer: 2.5 mu L; r end primer: 2.5. mu.L.

The PCR procedure was:

98℃，3min，×1；

(98℃、10s)→(60℃、20s)→(72℃、2min)，×40；

72℃，5min，×1。

the eGFP fluorescent protein gene amplification product after purification and enrichment is obtained by means of conventional agarose electrophoresis and gel cutting recovery in the prior art, and is sent to a third party company for sequencing to confirm that the recovered product is a target sequence.

S2: construction of expression vectors

The promoter of the lgals3bpb gene, the eGFP fluorescent protein sequence and pTAL2-cryaa vector (cat # 31823; see Darius Balciunas, Harnesing a high card go-capacity transcriptional for genetic applications in viral hybrids, PLoS Genet.2006 Nov; 2(11): e169.doi:10.1371/journal. pgen.0020169) were fused by homologous recombination to give pT2AL-cryaa-lgals3bpb-eGFP plasmid, the approximate process of which is shown in FIG. 2. The specific process is as follows:

using restriction enzymes Xma1 and Cla1 to carry out enzyme digestion treatment on pTAL2-cryaa vector, using DNA polymerase chain reaction to obtain an amplification product 1 of the Lgals3bpb gene promoter, and obtaining a PCR product A through purification and recovery; obtaining an amplification product 2 of the lgals3bpb gene promoter by using a DNA polymerase chain reaction, and obtaining a purified and recovered PCR product B; and amplifying the plasmid containing the eGFP fluorescent protein sequence by using a DNA polymerase chain reaction to obtain an eGFP sequence amplification product, and purifying and recovering to obtain a PCR product C. Then, the PCR product A, PCR product B and the PCR product C were ligated using homologous recombinase to form pT2AL-cryaa-lgals3bpb-eGFP plasmid, the schematic diagram of which is shown in FIG. 3.

The connecting body is: 2 × homologous recombinase: 10 mu L of the solution; cleaved pTAL2-cryaa vector: 100 mu g; PCR product A: 25 mu g of the mixture; PCR product B: 23 mu g of the solution; PCR product C: 30 μ g.

The connection time is as follows: 15 min; the temperature is as follows: at 50 ℃.

The plasmid obtained by construction is transformed into Escherichia coli DH5 competent cells by conventional means in the prior art. Respectively picking single clone into 40ml culture solution containing ampicillin, culturing at 37 ℃ and 180rpm for 12h, and purifying and extracting the plasmid by using an endotoxin-free plasmid extraction kit to obtain a plasmid vector pT2AL-cryaa-lgals3bpb-eGFP with high purity for removing substances such as escherichia coli cell bodies. The image of the colony screened by colony PCR is shown in FIG. 4.

S3: acquisition of transgenic zebrafish lines

Referring to fig. 5, the process of the step is as follows: pT2AL-cryaa-lgals3bpb-eGFP plasmid (40 ng/. mu.L) and transposase (100 ng/. mu.L, Tol2 transposase mRNA, whose template DNA sequence is shown in SEQ ID NO. 9) were ligated in the same manner as described in 1: 1 volume ratio, and injecting into zebra fish single cell fertilized egg (AB strain zebra fish, injection amount is 1nL), thereby randomly integrating the plasmid vector into the genome of zebra fish through the action of transposase. The transposon is a sequence capable of changing its position and comprises an intermediate sequence and a terminal directional repeat sequence, wherein the intermediate sequence can express transposase. Transposases recognize terminal repeats, which are then cleaved off and transferred to other locations in the genome. The transposase in the scheme is a tool for improving the plasmid insertion efficiency, and if the transposase is not used, the insertion of the foreign gene on the zebra fish genome can be carried out, but the efficiency is reduced.

Detecting the expression condition of a cryaa fluorescent protein signal in eyes under a fluorescent microscope 2 days after microinjection fertilization of the embryo of the single-cell fertilized egg, thereby determining whether microinjection is successful. Then, 3 days after fertilization, the expression and location of eGFP expression were examined under a fluorescent microscope to determine whether the recombinant plasmid was able to drive eGFP expression and specifically label microglia. Results of the observation of the crya-cerulean marker 3 days after fertilization are shown in FIG. 6 (crya-cerulean expresses blue fluorescence specifically in the lens of zebrafish and is commonly used to detect integration of plasmid into the chromosome after injection into zebrafish). Fertilized eggs with fluorescent expression were selected and cultured at a standard temperature of 28.5 ℃ until sexual maturity (F0 generation; founder). Mating the F0 generation with wild zebra fish to obtain fluorescent offspring individuals as F1 generations. The F1 generation was continued to be cultured until sexual maturity, and mated with wild type zebra fish to obtain fluorescent F2 generation. From this, the fluorescent line of zebrafish Tg (lgals 3bpb: eGFP) was constructed.

The whole-body eGFP signal of F1 (3dpf) zebra fish is detected, and the experimental result is shown in figure 7, and the eGFP signal is found to be specifically colonized in the brain and the skin. Since Langerhans cells are present on the skin, and are also tissue-resident macrophages, they share functional similarities with microglia. The lgals3bpb gene is involved in the immune process, so that the green fluorescent protein marker can also appear on the skin of the zebra fish. Langerhans cells and microglia are resident macrophages that belong to different tissues. If the promoter of the lgals3bpb gene is used to manipulate cells expressing the gene, in microglia, the manipulation of the gene will affect the brain microenvironment, such as neuronal development and function; in langerhans cells, these genetic manipulations are more likely to affect viral infections at their restricted sites and tumor-related immune processes. Therefore, the promoter of the lgals3bpb gene can promote the expression of effector genes in Langerhans cells and microglia, and the operation and experimental results of microglia cannot be influenced. In addition, if we use MTZ-NTR to specifically eliminate cells containing the promoter of the lgals3bpb gene, we can ensure that only microglia in brain are eliminated without affecting Langerhans cells by controlling the concentration of MTZ.

A zebrafish Tg (coro1a: DsRed) line was crossed with the aforementioned eGFP-expressing F0 generation (Tg (lgals 3bpb: eGFP)) to obtain a hybrid line Tg (coro1a: DsRed; lgals3bpb: eGPF). Referring to FIG. 8, when co 1a-DsRed + cells (red, left panel) and lgals3bpb-eGFP + cells (green, middle panel) were observed at 3dpf, a higher degree of overlap was found between the cells labeled with the promoter of the lgals3bpb gene and the cells labeled with the promoter of Coro1a gene (the fused image is right panel), indicating that the promoter of the lgals3bpb gene can drive the specific expression of the effector gene in microglia. Among them, the acquisition of the zebrafish Tg (coro1a: DsRed) line is described above in this example, except that the promoter and the effector gene are replaced with the promoter of the coro1a gene of the prior art (the promoter is disclosed in the documents Li, L., light imaging differentiation roles of macrophages and neutrophiles during purification tail fine regeneration.2012, Journal of Biological Chemistry 287,25353-25360.) and DsRed fluorescent protein. In FIG. 8, the arrow (arrowhead) indicates cells co-stained with DsRed and eGFP, and the arrow (arrow) indicates cells partially not co-stained (only DsRed signal). The promoter of Coro1a gene is mainly used for specifically labeling immune cells (leukocytes) including microglia, while the promoter of lgals3bpb gene is mainly used for labeling microglia. The immune cells of the brain are mainly microglia, so that the situation of coincidence of the labeled cells occurs. Cells labeled only with the promoter of Coro1a gene and not with the promoter of lgals3bpb gene were present in the fluorescence image. The inventors analyzed that the reason for this was that the cells marked by the promoter of these non-lgals 3bpb genes were not tissue-colonizing cells (i.e.non-microglia), but rather macrophages patrol to this location marked by the promoter of the Coro1a gene. The experimental result further indicates the specificity of the promoter marker of the lgals3bpb gene, and only marks microglia, but not marks other immune cells and neuron cells of the brain. We can use the characteristics of the promoter of the lgals3bpb gene to study the specificity of microglia and perform related operations.

The results of co-localization of the newly constructed transgenic line (fluorescent line of zebrafish Tg (lgals 3bpb: eGFP)) with the mesomeric microglia at the time of development up to 12 months are shown in FIG. 9 (red Lcp1 marker in the left panel, green eGFP marker in the middle panel, and fusion image of the two panels in the right panel). Anti-Lcp1 refers to an antibody against LCP1(lymphocyte cytosol protein 1). lcp1 and coro1a gene promoter are labeled cells, and the antibody is generally used for labeling the pan-microglia in the zebra fish brain sack. The above experimental results demonstrated that the expression of the effector gene controlled by the promoter of the coro1a gene was concentrated at the mesencephalon part-pan-microglia. In fig. 9, the arrow (arrowhead) indicates cells co-stained with Lcp1 and eGFP, and the arrow (arrow) indicates cells partially not co-stained (only Lcp1 signal). Adult zebrafish have macrophages of other sources to replace part of microglia in brain, so cells which do not express eGFP are available, and further, the specificity of the promoter marker of the lgals3bpb gene is demonstrated, and only the microglia is marked, but not other immune cells and neuronal cells.

Example 2: study of lgals3bpb gene and promoter thereof

(1) Study of expression Pattern of lgals3bpb Gene

As shown in FIG. 10, both apoeb gene (apolipoprotein E, accession No. NM-131098.2) and ccl34b.1 gene (chemokine (C-C motif) ligand 34b, duplicate 1, accession No. NM-001115054.2) are marker genes of microglia reported in the prior art, and are highly expressed in microglia. For the report of apoeb gene and ccl34b.1 gene as marker gene of Microglia, see the prior art documents (France sca Peri, Live Imaging of neural differentiation by Microglia Reveals a Role for v0-ATPase a1 In Phagosomal Fusion In Vivo, cell.2008May 30; 133(5):916-27.doi: 10.1016/j.cell.2008.04.037; Shuting Wu, Two phenolic and functional differentiation variants In adult zebraphis, Sci Adv.2020 Nov18; 6(47): eabd1160.doi: 10.1126/scd.1160.). The inventor discovers a plurality of genes which are enriched and expressed in microglia by means of gene sequencing and the like. The expression pattern of these genes with enriched expression was studied, and it was found that the expression pattern of lgals3bpb gene (accession No.: NM-212873.1, galectin 3binding protein) highly coincided with apoeb gene and ccl34b.1 gene, see FIG. 10.

In fig. 10, the coordinate axes represent gradients between data points, which are used to model their distance functions in high and low dimensional space. Where positive values of the gradient indicate attraction between data points in low dimensional space and negative values indicate repulsion. 0-6 in the right gradient plot represent the expression levels of the gene in different groups, and the expression levels are indicated by the shades of color. In fig. 10, the genes in the right box are the same as the expression patterns of apoeb gene and ccl34b.1 gene in immune cells of zebrafish brain, i.e. the enriched expression cell group is the same, including lgals3bpb gene, p2ry12 gene, vmp1 gene, fcer1gl gene, havcr1 gene, apoc1 gene, etc.

It is reported In the literature (France sca Peri, Live Imaging of neurological Degradation by Microglia Reveals a Role for v0-ATPase a1 In Phagosomal Fusion In Vivo, cell.2008May30; 133(5):916-27.doi:10.1016/j. cell.2008.04.037.) that In the transgene expression pattern of apoeb constructed by existing BACs, the effector Gene (GFP) carried by apoeb is not only expressed In Microglia, but also detected In brain neuron cells (immune cells of non-zebrafish brain) (see FIG. 11, which is derived from the paper of Pernce sca Percei mentioned above). In fig. 11, the thin arrows indicate microglia, the thick arrows indicate neuronal cells, and the neuronal cells are morphologically characterized by cells with long linear branches. This indicates that the use of apoeb gene to label microglia is not specific and can label neuronal cells, which is not conducive to the observation and manipulation of microglia. Therefore, some common microglia markers are only expressed in a large amount in microglia, but also have certain expression in other types of cells, particularly, apoeb genes can be simultaneously expressed in the microglia and the neuron cells, and thus serious interference is formed on the research of the microglia. As can be seen, the prior art is lack of genes (promoters) capable of specifically marking microglia, and the discovery of the expression mode of the lgals3bpb gene can overcome the defects of the prior art.

(2) Cloning of promoter of gene expressed in enriched microglia

Based on the genes selected in this example to be highly expressed in microglia, the inventors tried promoter capture of these genes to capture the promoter sequence of the labeled microglia. The identification of the core promoter of a gene is a relatively complex task, the sequence of the core promoter with the specificity of cellular expression is not known per se, and the position and length of the core promoter are difficult to predict before research findings. The core promoters of some genes are only a few hundred base pairs, and some are tens of thousands of base pairs. Some genes have their promoters closer to the coding sequence, some genes have their promoters farther from the coding sequence, and some genes have their promoters located upstream and some downstream of the coding sequence. These factors all hinder the discovery of the gene's promoter. The capture of the gene core promoter is shown in Table 1.

Table 1: microglial core promoter capture

A fluorescent image of microglia of transgenic zebra fish containing the vmp1 gene promoter is shown in FIG. 12 (3 days after fertilization), wherein the expression of DsRed is controlled by the promoter of the known coro1a gene, and the DsRed is expressed in large quantity, which indicates that the cells in the visual field are microglia (an immune cell), and in the cells, no eGFP signal appears, indicating that the upstream sequence of the initiation codon of the cloned vmp1 gene does not have the function of promoting the expression of the eGFP gene. Transgenic zebrafish studies were performed on the upstream sequence of the start codon of the havcr1 gene, as was the case for the vmp1 gene (see figure 13). The inventors analyzed the causes of the above phenomena: it is possible that the vmp1 gene promoter is not located 4000bp upstream of its start codon; or it may be that the promoter of the gene has been successfully cloned but is not effective in promoting expression of the effector gene in animal models. The above experimental results demonstrate that not arbitrary sequences having promoter effects can promote the expression of effector genes, i.e., that not arbitrary promoters are operable, and that cloning promoters and applying them to control the expression of effector genes in specific cells are difficult.

The function of the promoter of the Coro1a gene of the prior art is further explained with respect to the experimental results of FIGS. 13 and 14 as follows: the promoter of Coro1a gene can mark all immune cells (including those of macrophages, monocytes, granulocytes, lymphocytes, etc.), but based on the current belief that microglia are the only immune cells in the central nervous system region, we believe that the promoter of Coro1a gene marks pan-microglia (possibly including mature, immature and undifferentiated states) in the brain.

(3) Cloning of promoter of lgals3bpb Gene

The inventor firstly clones a sequence about 2000bp (SEQ ID NO.10) upstream of the initiation codon of the lgals3bpb gene, but cannot realize the effect of starting the expression of eGFP. Aiming at the lgals3bpb gene in the scheme, the high GC content of the promoter region and the repeated poly T reduce the amplification effect of the DNA polymerase chain reaction. So that the inventor can only amplify the gene by segments to finally obtain a 4141bp promoter sequence, and the sequence of the promoter sequence is shown in SEQ ID NO. 1. Amplification of the 4141bp sequence before the start codon of the lgals3bpb gene by DNA polymerase chain reaction initiated expression of the carried eGFP, and localization of eGFP-expressing cells to the brain (in combination with experimental data in example 1) indicated a specific marker for microglia, a novel finding.

In conclusion, the scheme provides an operable promoter sequence for marking the microglia, and the microglia can be operated through the specificity of the promoter. For example, the promoter carries a nitroreductase gene which can kill microglia particularly but not damage other macrophages; or a certain gene is supplemented or knocked out in microglia cells or Langerhans cells by utilizing the specificity of the promoter; fluorescent protein can also be integrated behind the promoter for positioning microglia, and the expression and the function of the gene (lgals3bpb gene) in the development process can be observed.

Example 3

The inventors also cloned the promoter of the apoc1 gene (SEQ ID NO.10) in example 2 using the procedure of example 1 and established a transgenic strain Zebra fish Tg (apoc 1: eGFP) using the procedure of example 1. The whole-body eGFP signal of F1 zebra fish is detected, the experimental result is shown in figure 14, the eGFP signal is specifically planted in the brain and the skin, and the yolk sac and the tail fin are marked by the promoter of the apoc1 gene besides the markers. As can be seen, the promoter signature of the apoc1 gene had a broader range than that of the lgals3bpb gene, and the promoter of the lgals3bpb gene had a relatively high specificity.

In addition, this example is identical to example 1 except that the promoter sequence is different and the corresponding cloning primer is different, but the green fluorescent protein controlled by the promoter of the lgals3bpb gene has stronger signal, which is more favorable for observing microglia.

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

SEQUENCE LISTING

<110> Chongqing Green Intelligent technology research institute of Chinese academy of sciences

<120> promoter of lgals3bpb gene and application thereof in construction of zebra fish model

<130> 2022.04.19

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 4141

<212> DNA

<213> Danio rerio

<400> 1

tgttgctctg aagtcagagg aggacgctgg gaaaatcgcc acctacattg tggcgtttac 60

ccaagatggc cgaattctgc tacctgaaac taagatcggg gatgtgaagt atgagggact 120

tgagttcatt tagtgtctgg acgtattttc atgccaagga ttggtaaatg ccttcttcta 180

ggtgatgtac atctagattt gggcattgta aggttaagaa taatgccaag tgtctgatgg 240

cagccaaaat gtggttgtaa atatcgactc ttaaaattaa agttgactct ttaattattt 300

taaatcacct gtcggagcct tcatcaaaag ttttttttat tgtttgcttc tctgtctttt 360

tcgaggttta gcgtttcacg ccagcactgt acttacagtg taatgaatgt aacagattgg 420

atgaagtgac agactcgaat agtttaccct acaaacatca ctgcagttgc catgtggttt 480

tgacgaatgt cttttttttt tttttactgt gaattgtttt gttttttact ttattgacaa 540

gtgtcttaaa cagatcagtt actttttaaa aaaaatctat aaatctgaac tgtcacaaaa 600

tgaacatcat gtttcatcga ttcaccagag ctatttattt gttgttgctg tattttgttt 660

ctgaagattg aaggttcata agaacggcac ttgtgtactc tggttatttc atccagttgt 720

ttgattcact cagttttgtt aaatcaaata tcacattaag ctaaatacac aggacagact 780

gcacccacag tgtggatgta cacagagttc atgaatttat tactcttaat cagtgtcttt 840

attgggtaca ccttactagt agcgagttga accccccttt gccttcagaa ctgccttaat 900

tctccgtggc ataaatattc ctcagagact ttggtccata ttgacattat agcatcatgc 960

atttgctgca gatttgttgg ctgcacatcc atgatgtgaa tctcccgttc caccacatcc 1020

caaaggtgct ctattggatt gagctctggt gactgtggag accatttgag taccgtgaac 1080

tcattgtcat gttcaagaaa ccagtctgag atgattcgga gatggttatc ctgctggaag 1140

tagatatcag aagatgggta cacagtggtc ataaagggat ggacattgtc ggcaacaata 1200

ctcaggtagg ctgtggcgtt gtcatgatgc tcagttggta ctaatgggcc caaagtgtgc 1260

caagacaata tccccatacc attacaccac caccacaagc ctgaaccgtt gatacaaggc 1320

tggatggatc catgctttta tgttgttgat gccaaattct gaccctacca ttcgaaaatc 1380

gcagcagaag tcgagactca tcagaccagg caacgtttgt ccaatcttct attgtccaat 1440

tttgctgagc ctgtgtgaat tgtagcctca gtttcctgtt cttagcggac aggattggca 1500

cccattgtgg tcttctgctg ctgtagccca tccgcctcaa ggttggatgt gttgtgtgtt 1560

cagagatgct tttctgcaga cctcggttgt agcgagtgca tatttgagtt actgttgcct 1620

ttctgtcagc tcaacccagt ctggccattc tcctctgacc tcttgtatta acaaggcatt 1680

tgcatccaca gaactgccgc accaacaacc ctgccacaat caaagttgct gccatgtgat 1740

tggctgatta gaaatttgca ttgagcattt tgacaggtgt gcctaataaa gtggctaggg 1800

agtgtatata taaacacaaa caaagacaaa attattagcc ctccttggaa atcataaatc 1860

cttttttaaa tatgtcctaa gagatgttta acagagccgt tacattttta tgatattttc 1920

tattatatga ttattcttta gaaagtcata tttcttttgg tttgtcttaa ataatagaat 1980

aaaaacaaaa agagtaaaaa ataaaggctg tgtttgaaca cgcctactac tcagtgggta 2040

ctacatttga atttgaattt actacctgac cattagaaaa gtaagatcta tacagtataa 2100

atgtgaatag tatgaatggt acttggacgt acaacagccg tcattttctg acctacccac 2160

atcagttggt cgcttcactc ccgttcatga attctctcac tgtgcattat gggatagcgt 2220

agcgtccatc tgatgtgcac ttcaaaatct agtcagaagt agtaggtcat ccgggtactt 2280

ctcgcataca gttttttatg aattctgtga atgaattcgg acatactact tgactcgcat 2340

actgttttta acttactata aagtatggaa gtatgcgatt tcggacgcag ctaaagtcaa 2400

taaaaattag cccctctttt ttcaaattgg aataaaaaaa ttgattgcct acagaacaat 2460

ccactgttgt ccaatgactt acctagttga cctaattaac ctagttaagt cttcaaattg 2520

cacttttagc tgaatattat tgtcacacaa tattgtgtac atcatggcaa agacgaaaga 2580

aattggttat tagaaatcag ttattaaaat ctgtatgttt aaaaatatgg ccaccacctg 2640

gtttttaccc atgctgtcct ccaaccccac aaacattatt atggtttaac agaagggtcc 2700

cagaaacata ctgtccctgg tagagatggg tcttcaaagt gccttgaagc tgaccagaga 2760

agaaaggaaa caaccccatg agtaaatgat gacagaattt tcaagtttta agtaaactat 2820

ccctgaacta ttgggtgaag acagcttgta actttttgaa ctcagttacc aaaatgtaga 2880

tcgggtttac tttaatctga aaagtaagta aaagttgtaa tatgttgcca agtatggtga 2940

cctatactaa taatttctgc tctgctttta acccatccaa gtacaaacac acaggagtaa 3000

acacaaacac ctgggaaaca attgcctaaa cagccactat gttaagaact aattacacca 3060

gatcaatcaa tgccttactc aagggaatca cctcagccat gtcattgagg gtggtaagtt 3120

accacctaca accctgacag gactcaaacc tgcaaccttc ttatcataag tccaacacta 3180

acccttagat ggtgtaagtg ttgtaggtgg tgggagagat caatcggtcc cttgctcaag 3240

gaactcacct cagccatggc attgagggtg gtgagttacc acctacaacc ctgacagaac 3300

ttgagcctct gcctttcgat cacaagtcca attctttaac cattaggcca cagctgcccc 3360

ttgtatggtg taagggttgt agctggtggg agagatcaat gggtcccttg ctcaaggaac 3420

tcatctcagc catggtattg agggtggtga gttaccacct acaaccctga cagaacttga 3480

gcctctgcct ttcgatcaca agtacaattc tttaaccatt aggccacagc tgccccttgg 3540

atggtgtaag ggttaaaggt ggtgggagag atcaatcagt gcctagctca agaaactcac 3600

ctcagccagg gtattgagag tggtgagtcc cttaagcaag gcaccgattg atctagtgta 3660

actagtactt cacatagtgg ctgttaatga aaccagacac agatgagact gactggaaac 3720

aaatgatcag gagcagcctc tgggaaaaca ggctctacag accccaggaa agcagaccta 3780

acccctattc tggcaattca cttgccagca aagagattca taagttatta tgggttatca 3840

atagggcaag tgactggtga ttgttttggg gaacactgta ttgtgggatt ttagtttgac 3900

taacacctca tgtggctaaa cttaacccag actttgctga caaacagttc ttatcgaaac 3960

ttcccaaatt gtacattttc atgaaacgaa acttttggtg tcctagaaat gtgattgcgc 4020

agcgtttcgt taaagtttca caattgcaaa cttgatttgg ccgttattta aaggaaagcc 4080

aagatgcttt gtaagtcatt cagttgtgaa ttgcatcctt gtcgttgctg tttctgacat 4140

c 4141

<210> 2

<211> 40

<212> DNA

<213> Artificial sequence

<400> 2

tcgaattcct gcagcccggg tgttgctctg aagtcagagg 40

<210> 3

<211> 40

<212> DNA

<213> Artificial sequence

<400> 3

gctgttgtac gtccaagtac cattcatact attcacattt 40

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

gtacttggac gtacaacagc 20

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence

<400> 5

tcctcgccct tgctcaccat gatgtcagaa acagcaacga 40

<210> 6

<211> 720

<212> DNA

<213> Artificial sequence

<400> 6

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

atggtgagca agggcgagga 20

<210> 8

<211> 41

<212> DNA

<213> Artificial sequence

<400> 8

tggatcatca tcgataggcc tttacttgta cagctcgtcc a 41

<210> 9

<211> 1950

<212> DNA

<213> Artificial sequence

<400> 9

atggaagagg tgtgcgactc ttccgccgca gcttcatcta ccgtccagaa ccagcctcag 60

gaccaagagc acccctggcc atatctgaga gagttcttct ccctgagcgg agtgaataag 120

gactcattta aaatgaagtg tgtgctgtgt cttccactca acaaagagat tagcgcgttt 180

aaatcctcac cctcgaacct gaggaagcac attgagcgta tgcaccctaa ctacctgaaa 240

aattactcca agctgaccgc ccagaagaga aagataggca ctagtactca cgcatccagc 300

agtaagcagt tgaaagtaga ctcagtgttc cctgttaagc atgtgtcccc tgtaacagtt 360

aacaaggcta ttctccggta cattatccaa ggtctgcacc cgttcagcac cgtcgaccta 420

ccctccttca aggagctgat tagtacgctt cagcctggaa ttagtgtgat tactcgccct 480

actctgagaa gcaaaatcgc agaagcggcg ctgatcatga aacagaaagt gactgctgca 540

atgagcgagg tggaatggat cgcgacgaca accgactgtt ggacagcacg taggaagtcc 600

tttataggcg tcactgccca ctggattaac cccggttcac tggagcgaca ctcagcagcc 660

ctggcctgca aacgccttat gggatctcac acttttgagg tgttagcgtc tgctatgaac 720

gacatccact cggaatacga aattagggac aaggtggtct gtaccacaac agactctgga 780

tctaacttta tgaaagcgtt tcgcgttttt ggcgtggaga acaatgatat cgaaacggag 840

gccagacgct gtgagtcaga cgacactgac agcgaaggct gcggcgaagg ttcagacggc 900

gtcgaatttc aggacgcgag tcgcgttctc gaccaggatg atggatttga atttcagtta 960

ccaaagcacc agaagtgtgc ctgtcatctt ctaaacctcg tctcttcggt cgacgcacaa 1020

aaagctctgt ccaacgagca ttacaagaag ctctaccggt ctgtcttcgg caagtgtcag 1080

gccctttgga acaagtcctc cagatccgct ctcgccgcag aggcggtaga gagtgagtcc 1140

agacttcagc tgctgagacc taatcagaca cgctggaaca gcactttcat ggcagtggac 1200

cgaatcttac aaatctgtaa agaagccggt gagggcgctc tccgcaacat ctgtacatca 1260

ctggaagtcc ctatgtttaa cccagctgag atgctcttct tgacagagtg ggctaacact 1320

atgcggccag tcgctaaagt cctcgacatc cttcaggccg aaaccaatac acagttaggc 1380

tggctccttc caagcgtgca ccaactcagt ctgaagctcc aaagactgca tcatagtctg 1440

cggtactgcg atcccctcgt ggatgctctg cagcagggaa tccagactcg gttcaaacac 1500

atgttcgaag accctgagat tatcgctgca gccatccttc tcccaaaatt tcgaacctcg 1560

tggacaaacg atgagaccat tattaagcgc ggcatggatt atatccgcgt ccaccttgaa 1620

cccctggacc acaaaaaaga gctggctaat tcaagtagtg acgatgagga tttctttgcc 1680

agcttgaagc cgacgacaca cgaggcctca aaggagctgg acggttacct ggcctgcgtc 1740

agcgatacaa gggagtcact gttgaccttc cctgcaatct gttcgctgtc gatcaagaca 1800

aacactccct tgcctgctag tgcagcgtgt gagagactct ttagcacagc aggactcctg 1860

ttcagcccta agagagcaag gcttgatacc aacaactttg agaaccagtt gcttctgaaa 1920

ctgaatttac gattttacaa cttcgagtga 1950

<210> 10

<211> 2023

<212> DNA

<213> Danio rerio

<400> 10

gtacttggac gtacaacagc cgtcattttc tgacctaccc acatcagttg gtcgcttcac 60

tcccgttcat gaattctctc actgtgcatt atgggatagc gtagcgtcca tctgatgtgc 120

acttcaaaat ctagtcagaa gtagtaggtc atccgggtac ttctcgcata cagtttttta 180

tgaattctgt gaatgaattc ggacatacta cttgactcgc atactgtttt taacttacta 240

taaagtatgg aagtatgcga tttcggacgc agctaaagtc aataaaaatt agcccctctt 300

ttttcaaatt ggaataaaaa aattgattgc ctacagaaca atccactgtt gtccaatgac 360

ttacctagtt gacctaatta acctagttaa gtcttcaaat tgcactttta gctgaatatt 420

attgtcacac aatattgtgt acatcatggc aaagacgaaa gaaattggtt attagaaatc 480

agttattaaa atctgtatgt ttaaaaatat ggccaccacc tggtttttac ccatgctgtc 540

ctccaacccc acaaacatta ttatggttta acagaagggt cccagaaaca tactgtccct 600

ggtagagatg ggtcttcaaa gtgccttgaa gctgaccaga gaagaaagga aacaacccca 660

tgagtaaatg atgacagaat tttcaagttt taagtaaact atccctgaac tattgggtga 720

agacagcttg taactttttg aactcagtta ccaaaatgta gatcgggttt actttaatct 780

gaaaagtaag taaaagttgt aatatgttgc caagtatggt gacctatact aataatttct 840

gctctgcttt taacccatcc aagtacaaac acacaggagt aaacacaaac acctgggaaa 900

caattgccta aacagccact atgttaagaa ctaattacac cagatcaatc aatgccttac 960

tcaagggaat cacctcagcc atgtcattga gggtggtaag ttaccaccta caaccctgac 1020

aggactcaaa cctgcaacct tcttatcata agtccaacac taacccttag atggtgtaag 1080

tgttgtaggt ggtgggagag atcaatcggt cccttgctca aggaactcac ctcagccatg 1140

gcattgaggg tggtgagtta ccacctacaa ccctgacaga acttgagcct ctgcctttcg 1200

atcacaagtc caattcttta accattaggc cacagctgcc ccttgtatgg tgtaagggtt 1260

gtagctggtg ggagagatca atgggtccct tgctcaagga actcatctca gccatggtat 1320

tgagggtggt gagttaccac ctacaaccct gacagaactt gagcctctgc ctttcgatca 1380

caagtacaat tctttaacca ttaggccaca gctgcccctt ggatggtgta agggttaaag 1440

gtggtgggag agatcaatca gtgcctagct caagaaactc acctcagcca gggtattgag 1500

agtggtgagt cccttaagca aggcaccgat tgatctagtg taactagtac ttcacatagt 1560

ggctgttaat gaaaccagac acagatgaga ctgactggaa acaaatgatc aggagcagcc 1620

tctgggaaaa caggctctac agaccccagg aaagcagacc taacccctat tctggcaatt 1680

cacttgccag caaagagatt cataagttat tatgggttat caatagggca agtgactggt 1740

gattgttttg gggaacactg tattgtggga ttttagtttg actaacacct catgtggcta 1800

aacttaaccc agactttgct gacaaacagt tcttatcgaa acttcccaaa ttgtacattt 1860

tcatgaaacg aaacttttgg tgtcctagaa atgtgattgc gcagcgtttc gttaaagttt 1920

cacaattgca aacttgattt ggccgttatt taaaggaaag ccaagatgct ttgtaagtca 1980

ttcagttgtg aattgcatcc ttgtcgttgc tgtttctgac atc 2023

<210> 11

<211> 4212

<212> DNA

<213> Danio rerio

<400> 11

cagttgtttg gactccttgc cctcaactgt ccacactata ttttataggg ccaaagagat 60

tgcaatttag taaataaaga ctgaggacga aaataataat aacatttatt acttgataaa 120

tgcttctgtt ggggcagcac ggtggcgcag tgggtagcac agtcggctcg cagcaagaag 180

gttgctagtt tgagccccgg ctgggtcagt tggcatttct gtgtggagtt tgcatgttcg 240

ccctatgttg gcgtaggttt cctcttggtg ctccggtttc gccctcagtc caaaatcatg 300

tggtacaggt gaattgggca agctaaattg tccgtcgtgt atgtgtagga atgagtgtgt 360

atggatgttt cccagtaatg ctgagtaaaa tatatgctgg ctaagttggc agttaattcc 420

gctgtggcaa caaatgaata aatactactg ttaatacaca caaacacaca cctacacaca 480

ggtttgtttt tgtgaatttg aggttttcat tgtttctata ctgtacaaac tgtattttca 540

ttgccctaca ccttacatga agctgtgtgc tgttttactt tcagaaataa aatcattata 600

cacgtcatta tacaagtcat tatacacatt tgcatcctaa tgtgacacaa aaacacgtac 660

atacacattt ttgtcctgat atgtcacaaa aaggcatact gtacattcag acacacatac 720

tgtatttata gctatggaaa aaagtttaag gtaaaaataa aaagttatgt gtaatttttg 780

ggtaattttg gttttattct gtaaactact aatgcctttt cttccaaacc cttttaggga 840

agagctttgc tcacaaaggt taaacaaaaa gcttgagatg gcacagcagg tcactgtcta 900

gactcataaa atgaaaaacc aagggccgtt gacttaacaa agtacataaa actaaaagaa 960

caacaggtga ctaattatgt gtgtgtgaga cgagctgttt gaatggcttt ggtcttaaag 1020

tgttctgcaa agcctgcaga taagtgacaa agggaacact ggaaggcccg tcctccacag 1080

actgcaagtg gtcagatgtg agcagaggta actgcagatc aatgcctctt ggtgtgatcc 1140

tctataaaca tcatgtcctc acggagcctc gcttcccctt attccagtct agacgctttc 1200

aacacttttc aagctgtcag cttttccgat cacgatcaag tcttttaata gatgcactat 1260

agtacaagaa agtattttgc aaaataaaga aaaatctatt aaacaggaaa aaaaatctcc 1320

ctgacctgag tgataaaaaa aataaacagg tcattgacca acaagaactg ctgattttgt 1380

gtgtgtgttt tgtgcccaca ctggattggg aaatacataa gatcaatgct gacaagatgt 1440

taactcttga ccttatttta aagtgtaaaa atgcaaatta cataaagtgc gatcaattat 1500

tatatgctac tataagatgc ttttttatta aataaattac tttgtcattt attgtttagt 1560

cttcatatca aacgttgatt tgaccctcaa aaacgtttat tttcaaaaag tccatacgtt 1620

gctctgatta tcacattatt ctgtcaatat ttttacattt tcatgattct ctgatgaaca 1680

aaatgttaaa ataacagcat atagaatgtt ttaatttcat ctaaaataat tttgtatttt 1740

aactgtttaa tctaccgcag gcaatattgc taggtagaaa caaaaacaat atgaccactt 1800

tcaaattcac aaaccttcat agcttcatgg ctgggacttt acataaatgt ctaatgtgtt 1860

tgaacattta agcacagaat atggcaaaaa atagtgtaca gcattgacga tgcagtgctt 1920

taattaaaat aaatcaaatg acaccatagg ccaatataag ataacatcta atgattctcc 1980

tgacctagaa tccaaaggtg gtttcaatgc agttagcaga atcctgcatg tatctatgtt 2040

tgtagacagc aggaataaag gttatgaaac tactaagaga taaacaactt caatttactt 2100

tagtccaaat ccagactctt tatctcatgt tgtgatgtgg ctggaaggtt aaagaactgt 2160

ggagtaaatc agtgtctcat gacattaaac aactacaaag ttatacaccc ttacaattgc 2220

gcagtcacaa ataataacaa cagtaataat aattttaaag ttgaattatg tttttgaaaa 2280

actaaggctg agattcaaga agtaggctat caatctcttt gctcgtgtgt tcagacatcc 2340

ataattcacc atatcaaaaa agaaacctgc atacagacag tgatcatttt gtatattaat 2400

aaaggagcag attcctaaaa agtgaccact aaagcgggtc acactttatt ttgataggtc 2460

gtttgttgaa tttatgttac attgcatcta catgccaact aattctcatt agattataag 2520

tagactgtta ggttggggtt agggtttgtt gacatgtact tgcaaaggtt cttatactca 2580

gttaaacgtt cttatataca gttaaatgtc tgttgaagga gcagtatcaa cagatattaa 2640

gcagtctatt aatactcaaa tggaccatca aaataaagtg ttaccctaaa gtgaatggtc 2700

agggggatac catgggatgg tgtaatatgc attttatttg tataaaatgc tgccatgtgc 2760

aatgtctgct acgggtcagt ttctttagtc actggtcaaa caagcagtat gaatgatcat 2820

tattgaactc tttatagata gtaatgaaat gtaaatgaaa acaaaaataa tttcttctca 2880

aaaattctgt tttacactgt aaacatgctg aggtccacac aattccttct tgttatccca 2940

acacaaactg attaagtaaa cttaattaaa tttacaaatg aaagtagctt gaacataaaa 3000

caattaagtt gtcccaaaaa aactcaggaa ttgtgttgat tcagctcatt ttagagtcat 3060

gattagtttg aacaagcagg aaaaatagtt ttgagcatac ataccagaat taactgtatc 3120

agtgaagtat gatgtatttt tatgccataa cactgatgct gtagtatatt tactagagga 3180

gatattacac aattgttttg tccttcagag tcatgtactg tagatgtaga aaaaagcacg 3240

taaccaactg taaaatccat gataataaat ggactttgta ccatatatgc aaggcaaggg 3300

taccagataa agaaagtacc cactttttta taacattgtt ttatttttga cagatgttgt 3360

ttcaaaggtc actggcactg aagtaaaata aagtagctca taacgaataa tcaaagaaaa 3420

tctccgtgaa aacagctgtt gtggcaaacg gagtttactt acctctaaag gatgtttact 3480

atagtcattt accacatcgc aaagctgtaa ggagaacgag agatgaacgc gaggaaagcg 3540

agtgattgca ggaggtcttg ataggtcaga ggtggtgtta ttacacaacg tcatctctgt 3600

gtccctataa agggacaagc catctgtggg acatcacaaa ctcagcccaa ttctttcgag 3660

gactgcgaag aggtacgcaa acttattttt gttgtggtca tttttcaagc agagcgaatc 3720

tagagagctt taacctgaaa cgacagaata acctttcttt attaggttca gtgcgcaaac 3780

acgcagtata ttggcgacat ctagccgcca tcccttgcta tgattggaaa atttagcata 3840

tttttgtaaa tgttagagga agataaataa aacactatta aacgtttaca aacaatgcgc 3900

aaacatattt ttaaaagcgc gaaatatata tttaacactt cattagaacc tttgtaataa 3960

attttaatct tatgtttatt gtttagtttt caaaagcaaa taacgtcttt gttttgatta 4020

atgacaaaaa tgattaatga caaaaaaatc ttcttgttca accagaagtt ttttttattt 4080

attttttatt ttttttaaga ttaacattag cgtaaaccat aaattgttta aataatatct 4140

taggaaataa tttaacaccg aatcggtcaa aattatgatg cctgagattt ctagctctct 4200

aacagtgatt cc 4212

Claims

1. The application of the promoter of the lgals3bpb gene in constructing a zebra fish model is characterized by comprising the following steps of:

2. The use of the promoter of lgals3bpb gene in constructing zebrafish model according to claim 1, wherein the primers for PCR reaction in S1 comprise:

F1-lgals3bpb：5’-tcgaattcctgcagcccgggTGTTGCTCTGAAGTCAGAGG-3’；

R1-lgals3bpb：5’-GCTGTTGTACGTCCAAGTACCATTCATACTATTCACATTT-3’；

F2-lgals3bpb：5’-GTACTTGGACGTACAACAGC-3’；

R2-lgals3bpb：5’-tcctcgcccttgctcaccatGATGTCAGAAACAGCAACGA-3’。

3. the use of the promoter of lgals3bpb gene in constructing zebrafish model according to claim 2, wherein the way to obtain F0 generation zebrafish in S3 is: simultaneously microinjecting the expression vector and the transposase into fertilized eggs of the zebra fish; selecting fertilized eggs expressing the fluorescent protein 3 days after fertilization; and culturing until sexual maturity to obtain F0 generation zebra fish.

4. The use of the promoter of lgals3bpb gene as claimed in claim 3, wherein the DNA template of transposase is represented by SEQ ID NO.9 in S3.

5. The use of the promoter of lgals3bpb gene in the construction of zebrafish model according to claim 4, wherein the culture temperature is 28.5 ℃ in S3.

6. The application of the promoter of the lgals3bpb gene in constructing a zebra fish model according to claim 5 is characterized in that in S3, zebra fish of F0 generation is mated with wild zebra fish, offspring expressing fluorescent protein is screened, and zebra fish of F1 generation is obtained; and mating the F1 generation zebra fish with wild zebra fish to obtain a stably inherited transgenic zebra fish strain.

7. The use of the promoter of lgals3bpb gene in the construction of zebrafish model according to claim 6, wherein in S1, the PCR reaction system comprises: 18. mu.L of water, 25. mu.L of buffer, 0.5. mu.L of DNA polymerase, 1.5. mu.L of template, 2.5. mu.L of forward primer and 2.5. mu.L of reverse primer.

8. The use of the promoter of lgals3bpb gene in the construction of zebrafish model according to claim 7, wherein the sequence of the fluorescent protein gene is shown in SEQ ID No.6 in S2.

A promoter of lgals3bpb gene, characterized in that its nucleotide sequence is shown in SEQ ID NO. 1.

10. The promoter of lgals3bpb gene according to claim 9, for controlling the expression of fluorescent protein in microglia of zebrafish.